Compositions and methods for osteogenic gene therapy

ABSTRACT

The present disclosure provides compositions and methods for increasing bone growth and/or enhancing wound healing, for example, fracture repair. The disclosure provides recombinant nucleic acids useful for promoting bone growth. For example, the disclosure provides recombinant nucleic acids that encode a fibroblast growth factor-2 (FGF-2) analog. The disclosure also provides vectors and cells incorporating these nucleic acids, as well as FGF-2 analogs encode by them. The disclosure also provides a mouse system of bone marrow transplantation and methods for producing as well as methods for using the system. Methods for inducing division and/or inducing differentiation of a hematopoietic stem cell are also provided, as are methods for enhancing bone growth and/or wound repair (for example, fracture repair).

CROSS REFERENCE TO RELATED APPLICATION

This is a divisional of U.S. patent application Ser. No. 12/879,842,filed Sep. 10, 2010, which is now U.S. Pat. No. 8,772,571, issued Jul.8, 2014, which is a divisional of U.S. patent application Ser. No.11/452,873, filed Jun. 13, 2006, which is now U.S. Pat. No. 7,816,140,issued Oct. 19, 2010, which claims benefit of priority to U.S. PatentApplication No. 60/690,696, filed Jun. 14, 2005. The entire disclosureof each of these applications are hereby expressly incorporated byreference in their entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made while one or more of the inventors were employedby the United States government (Department of Veterans Affairs) andwith United States government support: Assistance Award DAMD17-03-2-0021 from the United Sates Army Medical Research AcquisitionActivity and National Research Service Award NIAMSD Grant No.AR07543-14. The United States government has certain rights in theinvention.

FIELD

The present disclosure relates to the field of osteogenic therapy. Inparticular, the disclosure relates to methods of treating skeletaldisorders, such as osteoporosis and skeletal fractures with osteogenicgrowth factors, such as Fibroblast Growth Factor-2 and analogs thereof.

BACKGROUND

Growth factors, including members of the FGF and Wnt families, arepleiotropic regulators of proliferation, differentiation, migration, andsurvival in a variety of cell types. For example, basic fibroblastgrowth factor (bFGF or FGF-2) has been used to influence cellulargrowth, differentiation and migration (Bikfalvi et al., Endocrine Rev.18:26-45, 1997; Friesel et al., FASEB J. 9:919-925, 1995; Moyamoto etal., J. Cell. Physiol. 177:58-67, 1998; D'Amore et al., Growth Factors8:61-75, 1993) and is also a potent stimulator of angiogenesis (Moyamotoet al., J. Cell. Physiol. 177:58-67, 1998; D'Amore et al., GrowthFactors 8:61-75, 1993) and hematopoiesis in vivo (Allouche et al., Prog.Growth Factor 6:35-48, 1995). FGF-2 is involved in organogenesis(Martin, Genes Dev. 12:1571-1586, 1998), vascularization (D'Amore etal., Growth Factors 8:61-75, 1993), and wound healing (Ortega et al.,Proc. Natl. Acad. Sci. USA, 95:5672-5677, 1998), and plays an importantrole in the differentiation and/or function of various organs, includingthe nervous system (Ortega et al., Proc. Natl. Acad. Sci. USA,95:5672-5677, 1998), the skeleton (Montero et al., J. Clin. Invest.105:1085-1093, 2000), and several other organs (Bikfalvi et al.,Endocrine Rev. 18:26-45, 1997). Because of its angiogenic and anabolicproperties, FGF-2 has received considerable attention for potentialclinical applications, including wound healing and tissue repair.

The therapeutic utility of the FGF-2 protein therapy has been assessedin various animal models with promising results. Accordingly,administration of recombinant human FGF-2 protein improved the healingof ischemic wounds in rats (Quirinia et al., J. Plast. Reconstr. Surg.Hand Surg. 32:9-18, 1998), promoted scar-less healing of skin incisionalwounds in normal rats (Akasaka et al., J. Pathol., 203:710-720, 2004;Spyrou et al., Br. J. Plast. Surg. 55:275-282, 2002), enhanced woundhealing in healing-impaired diabetic rats (Takeuchi et al., J.Pharmacol. Exp. Ther. 281:200-207, 1997), and accelerated the woundhealing of chick embryo chorioallantoic membrane (Ribatti et al.,Angiogenesis 3:89-95, 1999). Administration of human recombinant FGF-2protein also promoted fracture healing in the monkey (Kawaguchi et al.,J. Clin. Endorinol. Metab. 86:875-880, 2001), improved cartilage repairin the rabbit (Tanaka et al., Tissue Engineering 10:633-641, 2004),stimulated early stages of tendon healing in the rat (Chan et al., ActaOrthop. Scand. 71:513-518, 2000), and led to formation of new trabeculaethat physically connect with pre-existing trabeculae in osteopenic rats(Lane et al., J. Bone Miner Res. 18:2105-2115, 2003; Lane et al.,Osteoporos Int. 14:374-382, 2003). Subcutaneous implantation ofcontrolled-release FGF-2 protein into the back of mice resulted in denovo formation of adipose tissue (Tabata et al., Tissue Engineering6:279-289, 2000).

Nonetheless, attempts to use FGF-2 in gene transfer approaches have ledto inconsistent results. While ex vivo FGF-2 promoted collateral vesseldevelopment in a rabbit hind limb ischemia model (Ishii et al., J. Vasc.Surg. 39:629-638, 2004) and improved blood flow and cardiac function ina swine myocardial ischemia model (Ninomiya et al., Gene Ther.10:1152-1160, 2003), in vivo expression of FGF-2 from a plasmid vectordid not significantly improve survival of rat ischemic myocutaneousflaps (Hijjawi et al., Arch. Surg. 139:142-147, 2004). Similarly, the invivo FGF-2 expression failed to preserve functional responses tophotoreceptor in a rat retinal degeneration model (Spencer et al., Mol.Ther. 3:746-756, 2001).

Thus, there exists a need for compositions and methods for increasingthe efficacy of treatment using osteogenic growth factors, such asFGF-2, to enhance bone growth and repair for the treatment of a widevariety of skeletal disorders. The compositions and methods disclosedherein address this need, providing numerous benefits, which will becomeapparent upon review of the specification.

SUMMARY

The present disclosure relates to compositions and methods forincreasing bone growth and/or enhancing wound healing, for example,fracture repair. Thus, a first aspect of the disclosure relates tomethods of inducing division and/or inducing differentiation of ahematopoietic stem cell. These methods involve expressing a heterologousnucleic acid that encodes an osteogenic growth factor in a stem cell,such as a hematopoietic stem cell. The growth factor is selected to i)promote self-renewal by the stem cell and ii) enhance bone growth invivo. Optionally, the growth factor increases angiogenesis in vivo.Examples of osteogenic growth factors include members of the fibroblastgrowth factor (FGF) family (for example, those FGF homologues that actvia FGF-receptors 1, 2 and/or 3, such as FF-1, FGF-2 and FGF-4), membersof the Wnt family, growth hormone, members of the angiopoietin familyand analogs thereof. Cells can be expanded in vitro, ex vivo or in vivo,for example, to enhance bone growth and/or wound repair.

In a particular example, hematopoietic stem cells or hematopoieticprogenitor cells are used as vehicles for delivering and expressingtherapeutic proteins that are osteogenic growth factors (such as FGF-2or therapeutic analogs thereof) to bone tissue. In certain embodiments,applicable to humans, the donor cells are CD34⁺ cells, which can beisolated from bone marrow, cord blood or peripheral blood. The selectedhematopoietic stems cells express CXCR4 receptors and have the abilityto home to, and to reside and engraft in the bone marrow space. Thesecells can be transduced with a therapeutic gene to target delivery of atherapeutic protein, such as a member of the FGF family, the Wnt familyor growth hormone, that promotes systemic and/or local bone formationand fracture healing. In a particular example, the therapeutic agent isan FGF-2 analog that promotes both bone formation and angiogenesis toassist in bone healing. This delivery system can be used to treat manytypes of systemic or local bone defects, such as bony defects frominjuries, fractures, or diseases like osteoporosis, osteomyelitis orcancer. Additional stem cells that express CXCR4 and that can target thebone marrow include endothelial stem cells and subsets of mesenchymalstem cells.

In disclosed embodiments, the therapeutic protein is expressed from anucleic acid construct delivered into the hematopoietic stem cell orhematopoietic progenitor cell. The cells are then delivered to targetsites, such as the endosteal bone surface of the bone marrow cavity,where they engraft and continue to produce progeny cells. Any suitablevector is used to deliver the nucleic acid construct into the cell.Promoters are chosen to drive the expression of the therapeutic protein,such as promoters that selectively drive expression in target tissuewhere expression is desired. For example, tissue-specific promoters areused that operate only in the presence of differentiated orundifferentiated osteoblasts. Inducible or suppressible promoters arealso used to regulate expression of the therapeutic protein in thetarget bone tissue.

The hematopoietic stem cells or progenitor cells that act as deliverycell vehicles can be isolated either from the bone marrow, peripheralblood, or umbilical cord blood of a subject or a donor (such as thesubject into whom the transduced cells will be transplanted), or theycan be derived from embryonic stem cells. In disclosed examples they areexposed to a nucleic acid construct carrying the nucleic acid sequencefor expressing the therapeutic protein which is constructed in a mannerthat it can enter the hematopoietic stem cells or progenitor cells (forexample using a viral vector or a nuclear localization sequence). Inparticular examples, the subject is pretreated in a manner that enhanceshoming of the delivery cells to the bone marrow. For instance, thesubject can be pre-treated with erythropoietin to induce the formationof red marrow throughout the skeleton, which leads to increased homingof the delivery cells to the skeleton. The transduced delivery cells canbe injected intravenously into the subject, and the cells then home tothe bone marrow and engraft there where they produce progeny cells thatin turn express the therapeutic protein. In particular examples, thetherapeutic protein is expressed in the endosteal bone niche of the bonemarrow site to induce bone formation by stimulating and recruitingnearby stromal cells in the marrow space to mature into osteoblasts,resulting in increased targeted bone formation.

Targeted delivery of the therapeutic protein, and/or therapeuticinduction of bone formation, can be more specifically directed byselecting a suitable promoter. For example when a promoter specific forerythroblasts is used, expression of the therapeutic protein isconcentrated in red marrow areas. Expression of the therapeutic proteincan be restricted to areas enriched with osteoblasts and/or itsprecursors by selecting a tissue specific promoter, such as an erythroidpromoter or an osteoblast promoter. Regulatable promoters (such as atetracycline regulatable system, e.g., Tet/on or Tet/off) can be used topermit therapy to be regulated by initiating or discontinuingadministration of tetracycline (or an analog thereof).

Another aspect of the disclosure relates to recombinant nucleic acidsuseful for promoting bone growth. For example, the disclosure providesrecombinant nucleic acids that encode a fibroblast growth factor-2(FGF-2) analog. The disclosure also provides vectors and cellsincorporating these nucleic acids, as well as FGF-2 analogs encoded bythem.

Another aspect of the disclosure relates to methods of producing a mousesystem of bone marrow transplantation involving transplantinghematopoietic stem cells or progenitor cells into sublethallyirradiated, myelosuppressed recipients. In one embodiment the donorcells are Sca-1⁺ cells isolated from bone marrow, cord blood orperipheral blood. Methods for identifying agents that modulate bonegrowth using the mouse bone marrow model are also disclosed.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a schematic illustrations of a chimeric FGF-2protein including a BVP2/4 secretion signal sequence (FIG. 1A) and theamino acid sequence surrounding the mature peptide cleavage site withinthe chimeric protein (FIG. 1B). FIG. 1A shows an exemplary chimeric(recombinant) human FGF-2 analog. The FGF-2 analog was constructed byadding the BVP2/3 secretion signal sequence to the 5′ end of the FGFgene. The positions of the four cysteines within the FGF-2 protein areindicated by inverted triangles. FIG. 1B represents the amino acidsequence surrounding the mature peptide cleavage site of the BMP2/4chimera (top; SEQ ID NO: 22) and that surrounding the mature peptidecleavage site of the BMP2/4-FGF-2 chimera (bottom; SEQ ID NO: 23). Thelast 5 amino acid residues of the C-terminus of the 284-residue BMP-2secretion signal sequence are shown in bold letters in FIG. 1B. The last16 residues of the C-terminal end of the BMP-4 secretion signal sequencethat had been incorporated in the BMP2/4 hybrid sequence are shown asitalic letters in FIG. 1B. The entire 8-residue propeptide of FGF-2(minus the start codon for methionine) was underlined in FIG. 1B. Therespective arrow denotes the cleavage site for mature BMP-4 and FGF-2protein, respectively. In the design of the BMP2/4-FGF-2 chimeric gene,the start codon for methionine of the FGF-2 gene was deleted andreplaced with the BMP2/4 secretion signal sequence.

FIGS. 2A and 2B are bar graphs demonstrating the effects of ex vivoadministration of pY-BMPFGFC2SC3N-transduced rat skin fibroblasts on theserum FGF-2 level (A) and the growth of implants (B) in a dorsal backimplant rat model. Gel-foam squares impregnated overnight with 4 millionprimary rat skin fibroblasts transduced with pY-β-gal, pY-FGF orpY-BMPFGFC2SC3N vectors were implanted subcutaneously into the dorsalback of syngeneic rats. Serum FGF-2 levels and size were evaluated 14days after implantation.

FIGS. 3A-3C are bar graphs comparing engraftment efficiency oftransplanted hematopoietic stem cells in mice under a variety ofconditions. FIG. 3A illustrates a comparison of engraftment efficiencyby the tail vein injection route and the retro-orbital injection routeof transplantation. The engraftment efficiency was assessed by measuringthe mean percent eGFP-positive cells in host mice peripheral blood overtime. Mice were transplanted with 400,000 Sca-1⁺ cells fromeGFP-transgenic donor mice through either the tail vein injection routeor the retro-orbital injection route. At indicated time points,peripheral blood was collected and assayed for percent eGFP cells byflow cytometry. FIG. 3B illustrates a comparison of the engraftmentefficiency in wild type C57BL/6J or W⁴¹/W⁴¹ recipient mice with orwithout sublethal irradiation. Engraftment was determined by measuringmean percent eGFP-positive cells in peripheral blood over time. Fivehundred thousand Sca-1⁺ cells from eGFP-transgenic donor mice weretransplanted into non-irradiated wild type (non-in wild), non-irradiatedW⁴¹/W⁴¹ (non-in W41/W41), irradiated wild type (irr wild) or irradiatedW⁴¹/W⁴¹ (in W41/W41) recipient mice At indicated time points, peripheralblood was collected and assayed for percent GFP cells by flow cytometry.FIG. 3C illustrates a comparison of the mean percent eGFP-positive cellsin peripheral blood of recipient mice preconditioned by sublethalirradiation either 4 hours or 24 hours prior to transplantation withSca-1⁺ cells from TgN-eGFP donors. Six W⁴¹/W⁴¹ recipient mice weresubjected to a single 500 cGy-dose of irradiation. Twenty hours later,another group of 6 W⁴¹/W⁴¹ recipient mice received an identical dose ofradiation. Four hours later, both groups were injected with 2×10⁶Sca-1⁺-enriched cells via retroorbital injection. At 12, 24, 36 and 52weeks post-transplantation, peripheral blood was collected and assayedfor percent GFP cells by flow cytometry (as an index of engraftment).All 12 mice were successfully engrafted.

FIGS. 4A and B are flow cytometry scatter plots. Percentage ofGFP-expressing donor cells in C57BL/6J Sca-1⁺ cells transduced with anHIV-based vector expressing the GFP marker gene five dayspost-transduction (left panel) and in peripheral blood of arepresentative recipient mouse nine weeks post transplantation (rightpanel). Percentage of GFP-expressing cells (GFP+ cells) was analyzed byFACS.

FIG. 4C is a bar graph comparing the mean percent eGFP-positive cellsperipheral blood of host mice transplanted with Sca-1⁺ cells transducedwith an HIV-based viral vector expressing the eGFP marker gene at 5, 9,11, and 22 weeks post transplantation.

FIGS. 5A and 5B are line graphs indicating serum FGF-2 and ALP levelswith respect to time post-transplantation (in weeks) in recipient mice.FIG. 5A illustrates serum FGF-2 levels at week 8, 10, 12, and 14post-transplantation were measured by ELISA according to Methods.Results are shown as mean±SEM (n=8 each). The FGF-2 group was the groupof mice receiving transplantation with pY-BMPFGFC2SC3N-transduced Sca-1+cells, whereas the GFP group was the group of recipient mice receivingtransplantation with pY-GFP-transduced Sca-1+ cells. *p<0.05 compared tothe GFP group. FIG. 5B illustrates the correlation between serum FGF-2levels and tibial extract ALP activity (normalized against dried boneweight) in recipient mice of Sca-1+ cells transduced with the modifiedFGF-2 MLV-vector (pY-BMPFGFC2SC3N) at 10- or 12-weekpost-transplantation.

FIG. 6 is a bar graph showing elevated serum PTH levels in recipientmice transplanted with pY-BMPFGFC2SC3N-transduced Sca-1+ cells. Serumparathyroid hormone (PTH) levels in mice transplanted with Sca-1+ cellstransduced with either pY-GFP (control) or pY-BMPFGFC2SC3N vector. Micetransplanted with Sca-1+ cells transduced with pY-BMPFGFC2SC3N vectorwere stratified according to serum FGF-2 levels: control (mean 35pg/ml); low (<200 pg/ml); intermediate (200-1000 pg/ml); and high (>1000pg/ml).

FIGS. 7A and 7B are schematic illustrations and a line graph,respectively, which illustrate inducibility of expression by the tet-onpromoter. FIG. 7A schematically illustrates a set of tet-on MLV-basedvectors expressing either eGFP or the modified FGF-2 gene, with orwithout the tet regulatable promoter. The MLV.FGF2* is the pY-modifiedFGF2 vector. The expression of the modified FGF2 is driven by the viralMLV-LTR promoter. The MLV.tet.eGFP and MLV.tet.FGF2* are doxycyclineinducible retroviral vectors expressing the eGFP or modified FGF-2 gene,respectively. In these two vectors, the expression of eGFP or FGF2 genewas driven by the TetO promoter, which is inducible doxycycline (atetracycline analog). The arrows indicate the direction of thetranscription. FIG. 7B illustrates the effects of doxycycline treatmenton the production of GFP protein in cells transduced with the pY.eGFPvector (closed symbols) and the pY.tet.eGFP (open symbols). Ten μl ofMLV.tet.eGFP viral stock was used to transduce 2×10⁵ HT1080 cells in6-well plates. An indicated amount of doxycycline (ranging from 0 to1000 ng/ml) was added to each well. Forty-eight hours aftertransduction, the cells were detached and the percentage ofGFP-expressing cells and the intensity of GFP expression were determinedby flow cytometry. The fold induction was calculated by comparing thevalue of mean intensity multiple by the percentage of the positivecells. The value from the control medium (without doxycycline) was setat 1.0-fold.

FIGS. 8A and 8B are bar graphs comparing FGF-2 levels and percent bonearea in young and old mice transplanted with Sca-1+ cells transducedwith MLV.β-gal or MLV.FGF2* vectors. Results are shown as mean±SD withn=9 or 10 per group.

FIG. 9 is a bar graph illustrating serum FGF-2 levels in recipient micetransduced with Sca-1+ cells expressing the β-gal control gene, the wildtype unmodified FGF-2 gene (WT FGF2), the modified FGF-2 gene with onlythe addition of the BMP2/4 propeptide (Pro FGF-2), and the modifiedFGF-2 gene with substitutions for C2 and C3 (Mutant FGF-2) and theBMP2/4 propeptide. Results are shown as mean±SD.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile, Annex C/St.25 text file, created on Sep. 10, 2010, 14,816 bytes,which is incorporated by reference herein. In the accompanying sequencelisting:

SEQ ID NO:1 is the nucleotide sequence of human FGF-2.

SEQ ID NO:2 is the amino acid sequence of human FGF-2.

SEQ ID NO:3 is the nucleotide sequence of an exemplary human FGF-2analog.

SEQ ID NO:4 is the amino acid sequence of an exemplary human FGF-2analog including a signal peptide (amino acid positions 1-284), an eightamino acid propeptide (italicized) and cysteine to serine substitutionat position 70 and a cysteine to arginin substitution at position 88 ofthe mature FGF-2 polypeptide.

SEQ ID NO:5 is the nucleotide sequence of the BMP2/4 signal sequence.

SEQ ID NO:6 is the amino acid sequence of the BMP2/4 signal sequence.

SEQ ID NOS:7-20 are the nucleotide sequence of oligonucleotide primers.

SEQ ID NO:21 is the nucleotide sequence of a Kozak initiation sequence.

DETAILED DESCRIPTION

Introduction

An important goal in the field of osteoporosis therapy is an osteogenictherapy which corrects the bone mineral deficit in patients withosteoporosis. One approach to developing osteogenic therapies hasfocused on gene therapy. However, such efforts have achieved onlylimited experimental success. To develop a successful therapy fortreating and inhibiting the development of osteoporosis and other bonedisorders, it is helpful to identify candidate genes that expressproteins to promote bone growth within bone tissue, and suitable cellsfor expressing them.

Factors to be considered in selecting a candidate gene include: theability to promote stem cell renewal; the ability to enhance bonegrowth. Additionally, the factors can promote angiogenesis. For example,bone morphogenetic proteins (BMPs) are able to promote enhanced bonegrowth in the short term, but cause stem cell differentiation that makesthem unsuitable for systemic therapies aimed at promoting growth andmaintenance of bone tissue over time. Rather, an osteogenic growthfactor that both promotes stem cell renewal and enhances bone growthprovides optimal therapeutic results, especially when systemic effectsare desirable. This disclosure provides methods for delivering suchosteogenic growth factors in vitro, ex vivo, and in vitro, to inducedivision and/or differentiation of stem cells, such as hematopoieticstem cells. In certain embodiments, the disclosed methods are used topromote localized and/or systemic bone formation, for example, toenhance bone strength and/or promote fracture repair.

Thus, one aspect of this disclosure concerns methods for inducingdivision and/or differentiation of stem cells, such as hematopoieticstem cells. The methods involve expressing a heterologous nucleic acidthat includes a polynucleotide sequence that encodes an osteogenicgrowth factor in a stem cell (and/or progenitor cell). In one aspect,these methods are useful for inducing bone formation in vitro, ex vivo,and in vivo. The selected cells are stem cells that are capable ofexpressing the therapeutic nucleic acid (or transgene), as well ashoming to and stably engrafting the bone marrow. Suitable donor cellsfor administration to a subject to promote increased bone growth arepluripotent stem cells, which are capable of self renewal, anddifferentiation along multiple lineage pathways. In specific examples,the stem cells are hematopoietic stem cells, such as embryonic stemcells or pluripotent hematopoietic stem cells. Various cells have beenreferred to as hematopoietic stem cells, including CD34 positive cellsisolated from the umbilical cord blood, the bone marrow, and theperipheral blood. Any one of these tissues provides a source of stemcells for use in the compositions and methods described herein, and suchstem cells can be isolated from these tissues using isolation proceduresknown in the art. One significant feature of these cells is that theyare able to home to, engraft, continue to produce progeny cells(including additional stem cells), and reside in the bone marrow cavity.The cells can also be any fraction of marrow stem cells, osteoblastcells and embryonic stem cells, so long as they are able to home to,reside and engraft in the bone marrow cavity. For example, endothelialstem cells and mesenchymal stem cells that express CXCR4 can besubstituted for hematopoietic stem cells in the methods disclosedherein.

A non-limiting example of such a stem cell is the Sca-1⁺ stem cellpopulation described below. For applications involving human subjects,it is useful to use a human hematopoietic stem cell, such as a humanbone marrow derived stem cell. Such stem cells can be isolated from thebone marrow, from umbilical cord vein blood or from the peripheralblood, for example, following treatment with Granulocyte-macrophagecolony-stimulating factor (GM-CSF) and or erythropoietin (EPO). Forexample, human bone marrow derived stem cells capable of homing to thebone marrow and giving rise to osteoblast lineage cells arecharacterized as CD34⁺, lin⁻ cells, more specifically CD34⁺, AC133⁺,lin⁻, CD45⁻, CXCR4⁺ (Kucia et al., Leukemia 19:1118-1177, 2005).Additionally, hematopoietic stem cells characterized as CD34⁺/CD38⁺cells (Chen et al., Stem Cells 15:368-377, 1997); CD73⁺, STRO-1⁺,CD105⁺, CD34⁻, CD45⁻, CD144⁻ cells (Tuli et al., Stem Cells 21:681-693);and CD29⁺, CD63⁺, CD81⁺, CD122⁺, CD164⁺, cMet⁺, bone morphogeneticprotein receptor 1B⁺, and neurotrophic tyrosine kinase receptor 3⁺ andCD34⁻, CD36⁻, CD45⁻, CD117⁻ (cKit⁻), and HLA-DR⁻ (D'Ippolito et al.,Journal of Cell Science 117:2971-2981, 2004) are also human pluripotenthematopoietic stem cells. Such cells are capable of expressingtherapeutic nucleic acids at appropriate levels, home to bone marrowthroughout the entire skeleton, undergo stem cell renewal underappropriate conditions (e.g., exposure to FGF), and give rise to cellsof the osteoblastic lineage. In certain embodiments, the hematopoieticstem cells express CXCR4, which facilitates homing to the bone marrow.Additionally, the stem cells can express one or more FGF receptors.

The hematopoietic stem cell can be a component of a population of cellsenriched in hematopoietic stem cells. For example, the population can beenriched by treating a population of cells with a growth factor thatpromotes expansion of hematopoietic lineage cells, such aserythropoietin (EPO), granulocyte colony stimulating factor (GCSF),and/or granulocyte macrophage colony stimulating factor (GMCSF). Thepopulation of cells can be treated either before or after isolation frombone marrow, cord blood or peripheral blood. Thus, the population ofcells can be enriched for hematopoietic stem cells by administering EPO,GCSF and/or GMCSF to a donor subject prior to isolating the populationof cells from bone marrow or peripheral blood. In some embodiments, thehematopoietic stem cells are obtained from the same subject to whom thecells are to be administered.

The osteogenic growth factor is selected to promote self-renewal by thestem cell and enhance bone formation in vivo. Optionally, the growthfactor also increases angiogenesis in vivo. Exemplary osteogenic growthfactors that promote stem cell self-renewal and that enhance boneformation include members of the FGF family of growth factors. Forexample, FGF family members that bind to and act via FGF-receptors 1, 2and/or 3, such as FGF-1, FGF-2, FGF-4, and analogs thereof, are used. Inother embodiments, members of the Wnt family of growth factors (such asWnts 1, 2, 2B, 3, 3B, 4, 5A, 5B, 6, 7A, 7B, 8A, 8B, 9A, 10A, 10B, 11 and16) are used to promote bone formation. In alternative embodiments, theosteogenic growth factor is selected from among growth hormone,angiopoietins 1-7 (e.g., Angptl2, Angptl3), glial cell nerve factor,stem cell factor, parathyroid hormone (PTH), insulin like growth factor(IGF), platelet derived growth factor (PDGF), vascular endothelialgrowth factor (VEGF), Cox-2, and TGF-β. Alternatively, nucleic acidsthat encode protein factors that regulate one of these genes, such astranscription factors, e.g., zinc finger binding proteins, can beintroduced into hematopoietic stem cells or progenitor cells anddelivered to the bone marrow to enhance bone growth and/or promotehealing of bone tissue.

The nucleic acids encoding osteogenic growth factors can be introducedinto the stem cells or progenitor cells as isolated linear nucleic acidsor incorporated into a wide variety of vectors, including plasmids,artificial chromosomes and viral vectors. The nucleic acids can beintroduced into the cells by a variety of means known in the art,including, for example, electroporation, lipid or liposome mediatedtransformation, biolistic (particle bombardment) transformation, andtransfection with a viral vector. The viral vector can be an integratingviral vector, such as a lentiviral vector, retroviral vector or anadeno-associated viral vector, or it can be a non-integrating viralvector, such as an adenoviral vector (e.g., a replication deficientadenovirus vector). Optionally, the nucleic acid can include a nuclearlocalization sequence. The nucleic acid typically includes atranscription regulatory sequence, including, for example, a promoter,to control expression of the nucleic acid (e.g., the therapeutic gene)to be expressed. The promoter can be a non-specific viral or non-viralpromoter, or it can be a tissue-specific promoter that operates in aselected subset of cells, such as erythroid precursors orundifferentiated or differentiated osteoblast lineage cells. Exemplarypromoters include erythroid specific promoters, such as the ankyrin-1promoter, an α-spectrin promoter, a ζ-globin enhancer and/or sequencesderived from the β-globin locus control region (LCR). Inducible orsuppressible promoters can also be used to regulate expression of theheterologous nucleic acid. Such promoters are typically ligand-dependenttranscription regulators and include, for example, such systems as theTet/on and Tet/off systems.

For ex vivo application, following introduction of the heterologousnucleic acid that encodes the osteogenic growth factor, thehematopoietic stem cell(s) and/or progeny thereof are introduced into asubject in need of bone formation. For example, subjects that canachieve improved health outcomes by increased bone formation includesubjects with osteoporosis and/or osteogenesis imperfecta, as well assubjects with bone fractures, including multiple severe fractures. Thetransduced cells can be introduced using any means available in the artfor transplanting bone marrow cells into a subject for the purpose ofsubsequent engraftment. For example, the transduced hematopoietic stemcells can be introduced into the peripheral circulation of a subject byinjection or transfusion. In the case of in vivo applications, nucleicacids encoding osteogenic growth factors can be introduced directly,either locally or systemically, into cells of a subject without anyintermediate step that involves isolation of the hematopoietic stemcells from a subject.

Thus, one aspect of the present disclosure relates to the use ofhematopoietic stem cells or hematopoietic progenitor cells expressingosteogenic growth factors for systemic or local skeletal administration,e.g., to the endosteal bone surface of the bone marrow cavity.Hematopoietic stem cells or progenitor cells have the unique property tohome to, reside, and engraft in the bone marrow space. Thus,hematopoietic stem or progenitor cells can be used to target delivery ofone or more therapeutic genes in the bone marrow to promote systemicand/or local bone formation and fracture healing. The methods describedherein can be used to treat bone diseases by expressing osteogenicgrowth factors in hematopoietic stem cells or hematopoietic progenitorcells, which are administered to a subject where they target bone tissueand promote skeletal growth and/or repair.

For example, one specific osteogenic growth factor for use in thedisclosed methods is FGF-2 (and analogs thereof). FGF-2 is known topromote stem cell renewal and is osteogenic. In addition, FGF-2increases angiogenesis, which is desirable to increase blood supply tonew bone. Thus, in one exemplary embodiment, the present disclosurerelates to compositions and methods employing FGF-2 and analogs thereofto promote systemic and/or local bone growth. Typically, the secretableanalog is selected to have increased stability of the FGF-2 polypeptideas compared to a wild-type FGF-2 polypeptide. Such analogs can includeat least one amino acid substitution that confers increased stability invitro, in vivo, or both in vitro and in vivo. For example, the FGF-2analog can include amino acid substitutions for cysteines at one or moreof amino acid positions 70 (C2) and/or 88 (C3). The substituted aminoacids can be nonglycosylatable or glycosylatable amino acids. Examplesof suitable substitutions for cysteines at positions 70 and/or 88include serine, asparagine and alanine. In one embodiment, thesecretable analog of FGF-2 is encoded by a nucleic acid that includes ina 5′ to 3′ direction: a polynucleotide that encodes a secretion signalsequence and a polynucleotide sequence that encodes a mature FGF-2polypeptide. For example, the secretion signal sequence can be a BMP2/4hybrid secreation signal sequence. The polynucleotide that encodes theFGF-2 analog can be operably linked to a constitutive (such as anon-viral or viral, e.g., CMV, promoter), tissue specific (includingerythroid specific and osteoblast specific promoters) or regulatablepromoter (for example, Tet-on and Tet-off). Optionally, the nucleic acidcan include additional polynucleotide sequences such vector sequences,and or additional sequences that facilitate secretion and/or processing,such as an FGF-2 propeptide encoding sequence. Such FGF-2 analogs andnucleic acids that encode them are also features of this disclosure.

Another aspect of the present disclosure relates to a new animal modelof bone marrow transplantation. The transgenic model system presentedherein overcomes many of the difficulties inherent in previous animalbone marrow transplantation models. The model system involvestransplantation and engraftment of donor hematopoietic stem cells and/orhematopoietic progenitor cells into an immunologically compatible,sublethally irradiated, myelosuppressed recipient mouse. The methodsinvolve transplanting at least about 100,000 donor cells, such as about400,000 donor cells per recipient.

For example, the donor cells can be pluripotent hematopoietic stem cellsor embryonic stem cells, such as Sca-1⁺ hematopoietic stem cells,isolated from bone marrow, spleen and/or peripheral blood of a donormouse. In some embodiments, the Sca-1⁺ hematopoietic stem cells areconstituents of a population of cells enriched for Sca-1⁺ cells. Thepopulation of cells can, for example, be enriched using magnetic beadsconjugated with an antibody specific for Sca-1.

The donor cells are genetically and/or phenotypically distinguishablefrom the recipient's cells. In certain embodiments, the donor cells arephenotypically distinguishable from the cells of the recipient. Forexample, the donor cells can express a transgene that encodes adetectable product or an enzyme that is capable of converting asubstrate into a detectable product. The detectable product can beoptically detectable, for example the optically detectable product canbe a green fluorescent protein (GFP), such as an enhanced GFP.

Typically, donor(s) and recipient(s) are immunologically compatible. Forexample, the donor and recipient mice can be syngeneic. To enhanceengraftment, the recipient mouse is genetically myelosuppressed. In anembodiment, the recipient mice have a mutation at the W locus (forexample, the recipient mice can be W⁴¹/W⁴¹ homozygous mice). Therecipient mice are typically conditioned with low levels of radiation ofbetween 50 and 1000 cGy, such as at least about 100 cGy, or at leastabout 200 cGy. In a specific example, the dose of irradiation isapproximately 500 cGy. For example the recipient mouse can be irradiatedusing a ⁶⁰Cobalt source and delivering from about 50 cGy to about 100cGy per minute (e.g., approximately 80 cGy) until the desired dose ofirradiation is obtained. Typically, the donor cells are transplantedafter a period of recovery. For example, the donor cells can betransplanted at least 2 hours, or at least 4 hours or up to about 24hours after sublethally irradiating the recipient mouse.

Engraftment is obtained by transplanting the donor cells into therecipient, e.g., via retroorbital injection. This approach yieldschimeric mice. When eGFP transgenic donor mice are utilized, GFPexpression can be used to monitor engraftment and to follow cell fate ofdonor cells, enabling monitoring and evaluation of stem cell expansionin recipient mice. Thus, in one specific embodiment, the disclosureprovides a W⁴¹/W⁴¹ mouse with a chimeric bone marrow that includeshematopoietic stem cells, hematopoietic progenitor cells and/orosteoblast lineage cells derived by descent from a donor cell thatexpresses an enhanced green fluorescent protein (eGFP).

Accordingly, this animal model provides an improved means of screeningfor genes, proteins, and/or other agents that stimulate stem cellrenewal and bone and other tissue regeneration. For example, methods foridentifying agents that modulate bone growth are provided. For example,the methods involve contacting one or more cells of a recipient mousewith a chimeric bone marrow with an agent, and detecting a change in oneor more indicator of bone growth. Exemplary indicators of bone growthinclude: hematopoietic stem cell survival, hematopoietic progenitor cellsurvival, osteoblast lineage cell survival, bone marrow engraftment(e.g., by a hematopoietic stem cell, a hematopoietic progenitor cell,and/or an osteoblast lineage cell), expression of one or more genes (inat least one of a hematopoietic stem cell, a hematopoietic progenitorcell and an osteoblast lineage cell), activity of one or more geneproduct (in at least one of a hematopoietic stem cell, a hematopoieticprogenitor cell and/or an osteoblast lineage cell), angiogenesis, bonemetabolism, bone mass, and bone density. By identifying a change in oneor more of these parameters an agent is identified that modulates bonegrowth.

In an embodiment, the cell is contacted with the agent by expressing atransgene, such as a transgene that encodes the agent or a transgenethat enzymatically produces the agent, in the cell. In anotherembodiment, the cell is contacted with a member of a library ofcompositions.

Additional details regarding the claimed compositions and methods areprovided throughout the disclosure. To facilitate review of the variousembodiments of this disclosure, the following explanations of specificterms are provided:

TERMS

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology may be found in Benjamin Lewin, Genes V,published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrewet al. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Additionally, numerical limitations given with respect toconcentrations or levels of a substance, such as a growth factor, areintended to be approximate. Thus, where a concentration is indicated tobe at least (for example) 200 pg, it is intended that the concentrationbe understood to be at least approximately (or “about” or “˜”) 200 pg.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” The abbreviation, “e.g.” is derived from the Latinexempli gratia, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

The term “gene” refers to a functional nucleic acid (e.g., DNA or RNA)sequence. A gene can include coding sequences necessary for theproduction of a functional RNA or polypeptide (e.g., a protein ofinterest). The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence so long as the desiredactivity or functional properties (e.g., enzymatic activity, ligandbinding, signal transduction) of the full-length polypeptide, orfragment, are retained. The term also encompasses sequences associatedwith (e.g., contiguous with or adjacent to) a coding region that areinvolved in regulation of expression of the coding sequence, such as 5′untranslated sequences including for example, a promoter, enhancers andother sequences which serve as the recognition sites for protein factorsinvolved in expression of the polynucleotide sequence. The term geneencompasses both cDNA (complementary DNA) and genomic forms of a gene.

A “transgene” is a heterologous nucleic acid, e.g., a heterologous“gene” introduced into a recipient cell or organism. Such a recipientcell, into which a heterologous nucleic acid has been introduced isreferred to as a “host” cell.

A “transformed” cell, or a “host” cell, is a cell into which a nucleicacid molecule (e.g., a transgene) has been introduced by molecularbiology techniques. A transformed cell or a host cell can be a bacterialcell or a eukaryotic cell.

A “population” of cells includes any number of cells. Thus, a populationof cells can include as few as one cell or can include many cells, forexample hundreds, thousands, hundreds of thousands or millions of cells.A “therapeutically effective” population of cells is a population ofcells sufficient to provide a desired effect following administration toa subject. The number of cells in a therapeutically effective populationof cells is dependent on a number of factors, including thecharacteristics of the cells, the genetics and/or physiology of thesubject to whom the cells are administered, the purpose of theadministration and the desired effect.

The terms “transduction,” “transfection” and “transformation” refer tothe introduction of heterologous DNA/RNA into cells. These terms areused interchangeably to refer to the introduction of nucleic acids intohost cells regardless of the methodology used. Common methods forintroducing nucleic acids into cells include calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, transfection with viral vectors, such asretroviral or adenoviral infection, and biolistics.

The term “nucleic acid” refers to a polymer of nucleotides of anylength. The term includes single- and double-stranded forms of DNA(deoxyribonucleic acid) and RNA (ribonucleic acid), as well as DNA-RNAhybrids. Generally, the term “nucleic acid” is synonymous with“polynucleotide” or “polynucleotide sequence,” unless clearly indicatedto the contrary. The repeating units in DNA (RNA) polymers are fourdifferent nucleotides, each of which comprises one of the four bases,adenine, guanine, cytosine and thymine (uracyl) bound to a deoxyribose(ribose) sugar to which a phosphate group is attached. Triplets ofnucleotides (referred to as codons) code for each amino acid in apolypeptide, or for a stop signal. The term codon is also used for thecorresponding (and complementary) sequences of three nucleotides in themRNA into which the DNA sequence is transcribed. Unless otherwisespecified, any reference to a DNA molecule is intended to include thereverse complement of that DNA molecule. Double-stranded DNA and RNA(dsDNA and dsRNA) have two strands, which can be defined with respect tothe products that they encode: a 5′→3′ strand, referred to as the plusor “sense” strand, and a 3′→5′ strand (the reverse compliment), referredto as the minus or “antisense” strand. Because RNA polymerase addsnucleic acids in a 5′→3′ direction, the minus strand of the DNA servesas the template for the RNA during transcription. Thus, the RNA formedhas a sequence complementary to the minus strand and identical to theplus strand (except that U is substituted for T). Except where singlestrandedness is required by context, DNA molecules, although written todepict only a single strand, encompass both strands of a double-strandedDNA molecule.

For convenience, short polynucleotides, typically of less than about 100nucleotides in length are often referred to as “oligonucleotides.”Similarly, very short polymers of two, three, four, five, or up to about10 nucleotides in length, can conveniently be referred to asdinucleotides, trinucleotides, tetranucleotides, pentanucleotides, etc.The nucleotides can be ribonucleotides, deoxyribonucleotides, ormodified forms of either nucleotide.

A “cDNA” or “complementary DNA” is a piece of DNA lacking internal,non-coding segments (introns) and transcriptional regulatory sequences.cDNA can also contain untranslated regions (UTRs) that are responsiblefor translational control in the corresponding RNA molecule. cDNA issynthesized in the laboratory by reverse transcription from messengerRNA extracted from cells.

A “recombinant” polynucleotide includes a polynucleotide that is notimmediately contiguous with one or both of the polynucleotide sequenceswith which it is immediately contiguous (one on the 5′ end and one onthe 3′ end) in the naturally occurring genome of the organism from whichit is derived. Thus, a recombinant nucleic acid can includepolynucleotide sequences that are “heterologous” with respect to eachother. A “heterologous” polynucleotide is a polynucleotide that is notnormally (e.g., in the wild-type genomic sequence) found adjacent to asecond polynucleotide sequence, or that is not normally found within aparticular cell, as the reference indicates. A heterologous nucleic acidor a heterologous polynucleotide can be, but is not necessarily,transcribable and translatable. In some embodiments, a heterologousnucleic acid is a cDNA or a synthetic DNA. In other embodiments, theheterologous polynucleotide sequence is a genomic sequence that encodesan RNA transcript. In other embodiments, a heterologous polynucleotideencodes a marker. Similarly, a recombinant protein is a protein encodedby a recombinant nucleic acid molecule. A recombinant protein can beobtained by introducing a recombinant nucleic acid molecule into a hostcell (such as a eukaryotic cell or cell line, such as a mammalian cellor yeast, or a prokaryotic cell, such as bacteria) and causing the hostcell to produce the gene product. Methods of causing a host cell toexpress a recombinant protein are well known in the art (see, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition,New York: Cold Spring Harbor Laboratory Press, 1989).

An “isolated” biological component (such as a nucleic acid or protein)has been substantially separated or purified away from other biologicalcomponents in the cell of the organism in which the component naturallyoccurs, such as, other chromosomal and extrachromosomal DNA and RNA, andproteins. Isolated nucleic acids and proteins include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.

The term “purified” refers to the removal of one or more extraneouscomponents from a sample. The term “purified” does not require absolutepurity; rather, it is intended as a relative term. For example, whererecombinant polypeptides are expressed in host cells, the polypeptidesare purified by, for example, the removal of host cell proteins therebyincreasing the percent of recombinant polypeptides in the sample.Similarly, where a recombinant polynucleotide is present in host cells,the polynucleotide is purified by, for example, the removal of host cellpolynucleotides thereby increasing the percent of recombinantpolynucleotide in the sample. Isolated polypeptides or nucleic acidmolecules, typically, comprise at least 50%, at least 60%, at least 70%,at least 80%, at least 90%, at least 95% or even over 99% (w/w or w/v)of a sample.

Similarly, the term “enriched” is a relative term, referring to aproportional increase in a constituent of a sample. For example, apopulation of cells is enriched for a particular component cell when thenumber of component cells is increased relative to the total number ofcells in a population. For example, the proportion of the component cellin a sample (e.g., as defined by a volume or total number of cells) canbe increased by 50% (such as from 10% to 15%) of the total number ofcells, or the increase can be more substantial. For example, theproportion of component cells can double or more than double (e.g.,increase by 2×, 3×, 4×, 5×, 10×, 20×, 50×, or more). Indeed in somecases, especially when dealing with a component cell that is present atvery low frequency in a starting sample, the proportion can increasehundreds of even thousands fold.

Cells, polypeptides and nucleic acid molecules are isolated by methodscommonly known in the art and as described herein. Purity of cells,polypeptides or nucleic acid molecules can be determined by a number ofwell-known methods, such as flow cytometry for cells, polyacrylamide gelelectrophoresis for polypeptides, or agarose gel electrophoresis fornucleic acid molecules.

A first polynucleotide sequence is “operably linked” to a secondpolynucleotide sequence when the first polynucleotide is in a functionalrelationship with the second polynucleotide. For instance, a codingsequence is operably linked to a transcription control sequence if thetranscription control sequence affects (e.g., regulates or controls) thetranscription or expression of the coding sequence. When recombinantlyproduced, operably linked polynucleotides are usually contiguous and,where necessary to join two protein-coding regions, are in the samereading frame. However, polynucleotides need not be contiguous to beoperably linked.

A nucleic acid that regulates the expression of a heterologouspolynucleotide sequence to which it is operably linked is referred to asan “expression control sequence” or a “transcription control sequence.”A transcription control sequence is operably linked to a nucleic acidsequence when the regulatory control sequence controls and regulates thetranscription and, as appropriate, translation of the nucleic acidsequence. Thus, transcription regulatory sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (typically, ATG) in front of a protein-encoding gene, splicingsignal for introns, maintenance of the correct reading frame of thatgene to permit proper translation of mRNA, and stop codons. The term“control sequences” is intended to include, at a minimum, componentswhose presence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

A “promoter” is a minimal sequence sufficient to direct transcription ofa nucleic acid. Also included are those promoter elements which aresufficient to render promoter-dependent gene expression controllable forcell-type specific, tissue-specific, or inducible by external signals oragents; such elements can be located in the 5′ or 3′ regions of thegene. Both constitutive and inducible promoters are included (see, e.g.,Bitter et al. Methods in Enzymology (1987) 153:516-544). For expressionin mammalian cell systems, promoters derived from the genome ofmammalian cells (for example, metallothionein promoter) or frommammalian viruses (for example, the cytomegalovirus immediate earlypromoter, the retrovirus long terminal repeat; the adenovirus latepromoter; the vaccinia virus 7.5K promoter) can be used. Alternatively,promoters that direct transcription in a selected tissue or set oftissues (tissue specific promoters) can be used. In one example,erythroid specific promoters that direct transcription in cells givingrise to red blood cells are used. Additionally, the promoter can be aregulatable promoter, such as a promoter that is regulated bytetracycline and/or analogs thereof. Promoters produced by recombinantDNA or synthetic techniques can also be used to provide fortranscription of the nucleic acid sequences.

“Expression” refers to transcription of a polynucleotide, and when usedin reference to a polypeptide, to translation. Expression is the processby which the information encoded by polynucleotide sequence is convertedinto an operational, non-operational or structural component of a cell.The level or amount of expression is influenced by cis-acting elementsand trans-acting binding factors, which are often subject to theinfluence of intra- and/or extra-cellular stimuli and signals. Theresponse of a biological system, such as a cell, to a stimulus caninclude modulation of the expression of one or more polynucleotidesequences. Such modulation can include increased or decreased expressionas compared to pre-stimulus levels. Expression can be regulated ormodulated anywhere in the pathway from DNA to RNA to protein (and caninclude post-translations modifications, e.g., that increase or decreasestability of a protein).

A polynucleotide sequence is said to “encode” a polynucleotide orpolypeptide if the information contained in the nucleotide sequence canbe converted structurally or functionally into another form. Forexample, a DNA molecule is said to encode an RNA molecule, such as amessenger RNA (mRNA) or a functional RNA (such as an inhibitory RNA(iRNA), small inhibitory RNA (siRNA), double stranded RNA (dsRNA), smallmodulatory RNA (smRNA), antisense RNA (asRNA) or ribozyme, if the RNAmolecule is transcribed from the DNA molecule, and contains at least aportion of the information content inherent in the DNA molecule. A DNAor RNA molecule is said to encode a polypeptide, e.g., a protein, if theprotein is translated on the basis of a sequence of trinucleotide codonsincluded within the DNA or RNA molecule. Where the coding molecule is aDNA, the polypeptide is typically translated from an RNA intermediarycorresponding in sequence to the DNA molecule.

The term “polypeptide” refers to any chain of amino acids, regardless oflength or post-translational modification (for example, glycosylation orphosphorylation), such as a protein or a fragment or subsequence of aprotein. The term “peptide” is typically used to refer to a chain ofamino acids of from about 3 to about 30 amino acids in length.

A “vector” is a nucleic acid as introduced into a host cell, therebyproducing a transformed host cell. Exemplary vectors include plasmids,cosmids, phage, animal and plant viruses, artificial chromosomes, andthe like. Vectors also include naked nucleic acids, liposomes, andvarious nucleic acid conjugates. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(for example, vectors having a bacterial origin of replication replicatein bacteria hosts). Other vectors can be integrated into the genome of ahost cell upon introduction into the host cell and are replicated alongwith the host genome. Some vectors contain expression control sequences(such as, promoters) and are capable of directing the transcription ofan expressible nucleic acid sequence that has been introduced into thevector. Such vectors are referred to as “expression vectors.” A vectorcan also include one or more selectable marker genes and/or geneticelements known in the art.

A “marker” is a molecule that serves to distinguish one cell fromanother. In the context of the present disclosure, a marker serves as anindicator of cell origin unless otherwise indicated. Typically, a markeris selected for ease of detection, e.g., by optical means. Commonmarkers include fluorescent proteins, such as green fluorescent protein(GFP) and numerous variants thereof. Other markers include proteins withenzymatic activities that convert a fluorogenic or chromogenic substrateinto a fluorescent or visible product, or that convert an isotopicallylabeled substrate into a radioactive product. Examples of such enzymaticmarkers include firefly luciferase, chloramphenicol acetyltransferase(CAT), β-glucuronidase and β-galactosidase. A polynucleotide encoding amarker can be operably linked to a transcription control sequence andintroduced into cells. Markers also include selectable markers, theactivity of which can be measured as relative resistance or sensitivityto a selection agent, such as an antibiotic. Naturally occurring markersinclude readidly detectable traits such as coat (fur) color, polymorphicantigens, and the like. Alternatively, the marker can be a geneticmarker such as a nucleic acid polymorphism.

A “stem cell” is an undifferentiated cell, capable of indefiniteproliferation and generation of differentiated progeny cells. The term“progeny” of a cell or “progeny cell” refers to a cell generated by oneor more cycles of DNA replication and division of a parental cell. Inmammalian cells, for example, a single cycle of replication and divisiontypically gives rise to two progeny cells. Subsequent cycles ofreplication and division give rise to exponentially increasing numbersof progeny cells from a single parental or progenitor cell. The progenyof a stem cell can include additional stems cells. This process isreferred to as “self-renewal.” Progeny of a stem cell can also includecells of one or more cell lineages or differentiated phenotypes, e.g.,hematopoietic cells or osteoblast lineage cells.

Stem cells can be divided into three broad categories on the basis ofthe variety of differentiated progeny generated by the stem cell.“Totipotent” stem cells, e.g., blastomeres, can give rise to every celltype of an organism. “Pluripotent” stem cells give rise todifferentiated cells of any of the three germ layers. “Multipotent” (or“unipotent”) stem cells give rise to a limited set of cell types,typically restricted to a single tissue or lineage. Stem cells can bederived or obtained from either embryonic or adult organisms. Embryonicstem (“ES”) cells are cells obtained from the inner mass cells of ablastocyst. The term “adult” stem cell refers to undifferentiated cellswithin a specific tissue, which can be found in and obtained or derivedfrom either adult or immature, including embryonic organisms that haveundergone sufficient organogenesis that distinct multicellular tissuesand organs can be identified. Such adult stem cells are oftenmultipotent cells.

The term “hematopoietic stem cell” refers to a heterogenous class ofcells typically isolated from bone marrow, cord blood, peripheral bloodor embryonic liver. Hematopoietic stem cells can be either pluripotentor multipotent stem cell that gives rise to hematopoietic cells, thatis, red and white blood cells. Additionally, certain pluripotenthematopoietic stem cells give rise to cells of various lineages and germlayers, including one or more of muscle lineage cells, neural lineagecells and osteoblast lineage cells.

The term “osteoblast” includes bone progenitor cells which have thecapacity to form, or to contribute to the formation of, new bone tissue.Osteoblasts include osteocytes and more immature osteoblast lineagecells. The term “cell lineage” refers to the ancestry (that is, theprogenitor cell and program of cell divisions) of a cell. Thus, an“osteoblast lineage cell” is any cell that arises by division of acommitted osteoblast progenitor, such as a preosteoblast, osteoblast orosteocyte.

The term “osteogenic” in reference to an agent, indicates that the agentinduces, promotes or otherwise facilitates bone growth (e.g., new boneformation), maintenance and/or repair. “Bone formation” is a measurableproperty of bone (skeletal) tissue. Thus, bone formation includes one ormore of an increase in cellularity (e.g., increase in the number,survival and/or longevity of hematopoietic stem cells, hematopoieticprogenitor cells and/or osteoblast lineage cells) of the bone marrow, anincrease in bone mass, and/or an increase in bone density. Boneformation can be monitored or evaluated by measuring (directly orindirectly) one or more indicators of bone growth such as: hematopoieticstem cell survival, hematopoietic progenitor cell survival, osteoblastlineage cell survival, bone marrow engraftment by at least one of ahematopoietic stem cell, a hematopoietic progenitor cell, and anosteoblast lineage cell, expression of one or more genes in at least oneof a hematopoietic stem cell, a hematopoietic progenitor cell and anosteoblast lineage cell, activity of one or more gene product (e.g.,skeletal alkaline phosphatase) in at least one of a hematopoietic stemcell, a hematopoietic progenitor cell, angiogenesis, bone metabolism,bone mass, and bone density.

In the context of this disclosure, a growth factor is a peptide orpolypeptide agent that promotes division (e.g., replication,proliferation) and or differentiation of one or more cells. Anosteogenic growth factor is a growth factor that promotes division,differentiation or both division and differentiation, of a progenitorcell into progeny cells that contribute directly or indirectly to boneformation.

The term “mammal” includes both human and non-human mammals. Similarly,the term “subject” or “patient” includes both human and veterinarysubjects or patients.

Therapeutic Nucleic Acids

In one aspect, the present disclosure relates to nucleic acids thatencode therapeutic transgenes suitable for administration for promotingbone growth (for example, bone formation) in humans and other mammals.The nucleic acids encode osteogenic growth factors that are capable ofpromoting stem cell renewal, increasing bone formation and, in somecases, enhancing angiogenesis. Exemplary osteogenic growth factors thatpromote stem cell self-renewal and that enhance bone formation includemembers of the FGF family of growth factors. For example, FGF familymembers that bind to and act via FGF-receptors 1, 2 and/or 3, such asFGF-1, FGF-2, FGF-4, and analogs thereof, are osteogenic growth factors.Members of the Wnt family of growth factors (such as Wnts 1, 2, 2B, 3,3B, 4, 5A, 5B, 6, 7A, 7B, 8A, 8B, 9A, 10A, 10B, 11 and 16) are alsoamong the osteogenic growth factors suitable for use in the methodsdescribed herein. Additionally, growth hormone, angiopoietins 1-7 (e.g.,Angptl2, Angptl3), glial cell nerve factor, stem cell factor,parathyroid hormone (PTH), insulin like growth factor (IGF), plateletderived growth factor (PDGF), vascular endothelial growth factor (VEGF),Cox-2, and TGF-β are exemplary osteogenic growth factors. Thus, thetherapeutic nucleic acids include polynucleotide sequences that encodeat least one osteogenic growth factors.

One specific example of an osteogenic growth factor is fibroblast growthfactor-2 (FGF-2). The use of FGF-2 serves as an example throughout thisdisclosure. Nonetheless, one of skill in the art will recognize that anyof the growth factors listed above, and indeed any growth factor thatsatisfies the criteria of promoting self-renewal of hematopoietic stemcells, enhancing bone formation in vivo, and optionally, promotingangiogenesis in vivo is a suitable osteogenic growth factor in themethods disclosed herein.

The nucleotide and amino acid sequences of human FGF-2 are representedby SEQ ID NO:1 and SEQ ID NO:2, respectively. While the compositions andmethods are described with respect to the human FGF-2 homolog, which isparticularly suited for administration to human subjects, thecompositions and methods disclosed herein are equally applicable toother mammalian FGF-2 orthologs, which can be selected by one of skillto correspond to the subject to which the nucleic acid, protein or cellis to be administered. Thus, for example, if the subject is a domesticlivestock animal, such as a cow, a pig or a sheep, the FGF-2 nucleicacid can be selected from nucleic acids represented by GENBANK®accession nos: AX085265, AJ577089, and NM_001009769, respectively.Similarly, suitable FGF-2 homologs can be selected, and analogsproduced, corresponding to any species of interest.

Exemplary analogs include modified FGF-2 nucleic acids and proteins thathave been modified to include a signal peptide that promotes secretionof the translated FGF-2 product. One suitable secretion signal sequenceis a hybrid BMP2/4 secretion signal sequence as represented by SEQ IDNO: 5 (nucleotide) and SEQ ID NO:6 (amino acid), which facilitatessecretion of the translated product. The nucleotide and amino acidsequences of an exemplary FGF-2 analog are provided in SEQ ID NO:3 andSEQ ID NO:4, respectively. A FGF-2 analog can include one or more aminoacid substitutions (or additions or deletions) that increases stabilityof the secreted protein, typically without altering its activity. Forexample, one or more cysteine residues (up to all four of the cysteineresidues) can be modified as shown in the analog schematicallyillustrated in FIG. 1A. Typically, the second and third cysteines, thatis, cysteines at positions 70 and 88 are mutated. For example, suitablemutations include cysteine to serine substitutions and cysteine toasparagine substitutions.

Therapeutic nucleic acids encoding a growth factor suitable forincreasing bone growth, such as FGF-2 or an analog thereof includedeoxyribonucleotides (DNA, cDNA) or ribodeoxynucletides (RNA) sequences,or modified forms of either nucleotide, which encode the fusionpolypeptides described herein. The term includes single and doublestranded forms of DNA and/or RNA.

Polynucleotide sequences described herein include polynucleotidesequences, such as the sequences represented by SEQ ID NO:1 and 3, whichencode FGF-2 and an exemplary analog, as well as polynucleotidesequences complementary thereto. For example, a polynucleotide thatencodes a modified FGF-2 amino acid sequence represented by SEQ ID NO:4is a feature of this disclosure.

In addition to the polynucleotide sequences represented by SEQ ID NOS:1and 3, and the amino acid sequences represented by SEQ ID NOS:2 and 4,polynucleotide and amino acid sequences that are substantially identicalto these polynucleotide sequences can be used in the compositions andmethods of the disclosure. For example, a substantially identicalsequence can have one or a small number of deletions, additions and/orsubstitutions. Such nucleotide and/or amino acid changes can becontiguous or can be distributed at different positions within thenucleic acid or protein. A substantially identical sequence can, forexample, have 1, or 2, or 3, or 4, or even more nucleotide or amino aciddeletions, additions and/or substitutions. Typically, the one or moredeletions, additions and/or substitutions do not alter the reading frameencoded by a polynucleotide sequence, such that a modified (“mutant”)but substantially identical polypeptide is produced upon expression ofthe nucleic acid.

The similarity between polynucleotide and/or amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity); the higher thepercentage, the more similar are the primary structures of the twosequences. Thus, a polynucleotide that encodes FGF or an FGF-2 analog(or another osteogenic growth factor) can be at least about 95%, or atleast 96%, frequently at least 97%, 98%, or 99% identical to SEQ ID NO:1(or SEQ ID NO:3 or the corresponding growth factor encodingpolynucleotide) or to at least one subsequence thereof. Methods ofdetermining sequence identity are well known in the art. Variousprograms and alignment algorithms are described in: Smith and Waterman,Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch J. Mol. Biol. 48:443,1970; Higgins and Sharp Gene 73:237, 1988; Higgins and Sharp CABIOS5:151, 1989; Corpet et al. Nucleic Acids Research 16:10881, 1988; andPearson and Lipman Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul etal. Nature Genet. 6:119, 1994 presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Thus, a sequence (that is a polynucleotide or polypeptide sequence) thatis substantially identical, or substantially similar polynucleotide to apolynucleotide of SEQ ID NO:1, 3, or 5 (or to a polypeptide sequence ofSEQ ID NO:2, 4 or 6) is encompassed within the present disclosure. Suchpolynucleotides can include, e.g., insertions, deletions, andsubstitutions relative to any of SEQ ID NOs:1, 3 and/or 5. For example,such polynucleotides are typically at least about 70% identical to areference polynucleotide (or polypeptide) selected from among SEQ IDNO:1 through SEQ ID NO:5. That is, at least 7 out of 10 nucleotides (oramino acids) within a window of comparison are identical to thereference sequence selected SEQ ID NO:1-5. Frequently, such sequencesare at least about 80%, usually at least about 90%, and often at leastabout 95%, or more identical to a reference sequence selected from SEQID NO:1 to SEQ ID NO:5. For example, the amino acid or polynucleotidesequence can be 96%, 97%, 98% or even 99% identical to the referencesequence, e.g., at least one of SEQ ID NO:1 to SEQ ID NO:5

Another indicia of sequence similarity between two nucleic acids is theability to hybridize. The more similar are the sequences of the twonucleic acids, the more stringent the conditions at which theyhybridize. Substantially similar or substantially identical nucleicacids to SEQ ID NO:1 and 3 (and to subsequences thereof) include nucleicacids that hybridize under stringent conditions to any of thesereference polynucleotide sequences. Thus, a nucleic acid that hybridizesunder stringent conditions to a reference polynucleotide sequenceselected from among SEQ ID NOs:1 and 3 is substantially identical orsubstantially similar to the polynucleotides encoding FGF-2 and/or ananalog thereof.

The stringency of hybridization conditions are sequence-dependent andare different under different environmental parameters. Thus,hybridization conditions resulting in particular degrees of stringencyvary depending upon the nature of the hybridization method of choice andthe composition and length of the hybridizing nucleic acid sequences.Generally, the temperature of hybridization and the ionic strength(especially the Na⁺ and/or Mg⁺⁺ concentration) of the hybridizationbuffer determine the stringency of hybridization, though wash times alsoinfluence stringency. Generally, stringent conditions are selected to beabout 5° C. to 20° C. lower than the thermal melting point (T_(m)) forthe specific sequence at a defined ionic strength and pH. The T_(m) isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe. Conditionsfor nucleic acid hybridization and calculation of stringencies can befound, for example, in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001,NY; Tijssen, Hybridization With Nucleic Acid Probes, Part I: Theory andNucleic Acid Preparation, Laboratory Techniques in Biochemistry andMolecular Biology, Elsevier Science Ltd., NY, 1993, and Ausubel et al.Short Protocols in Molecular Biology, 4^(th) ed., John Wiley & Sons,Inc., 1999.

For purposes of the present disclosure, “stringent conditions” encompassconditions under which hybridization occurs only if there is less than25% mismatch between the hybridization molecule and the target sequence.“Stringent conditions” can be broken down into particular levels ofstringency for more precise definition. Thus, as used herein, “moderatestringency” conditions are those under which molecules with more than25% sequence mismatch do not hybridize; conditions of “mediumstringency” are those under which molecules with more than 15% mismatchdo not hybridize, and conditions of “high stringency” are those underwhich sequences with more than 10% mismatch do not hybridize. Conditionsof “very high stringency” are those under which sequences with more than6% mismatch do not hybridize. In contrast nucleic acids that hybridizeunder “low stringency conditions include those with much less sequenceidentity, or with sequence identity over only short subsequences of thenucleic acid.

For example, in nucleic acid hybridization reactions, the conditionsused to achieve a particular level of stringency varies depending on thenature of the nucleic acids being hybridized. The length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA versus DNA) of thehybridizing regions of the nucleic acids all influence the selection ofappropriate hybridization conditions. Additionally, whether one of thenucleic acids is immobilized, for example, on a filter can impact theconditions required to achieve the desired stringency.

A specific example of progressively higher stringency conditions is asfollows: 2×SSC/0.1% SDS at about room temperature (hybridizationconditions); 0.2×SSC/0.1% SDS at about room temperature (low stringencyconditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringencyconditions); and 0.1×SSC at about 68° C. (high stringency conditions).One of skill in the art can readily determine variations on theseconditions (e.g., with reference to Sambrook, Tjissen and/or Ausubel,cited above). Washing can be carried out using only one of theseconditions, e.g., high stringency conditions, or each of the conditionscan be used, e.g., for 10-15 minutes each, in the order listed above,repeating any or all of the steps listed. However, as mentioned above,optimal conditions vary, depending on the particular hybridizationreaction involved, and can be determined empirically.

Additionally, the nucleic acid encoding the osteogenic growth factorpolypeptides can also include polynucleotide sequences, such asexpression regulatory sequences and/or vector sequences that facilitatethe expression or replication of the nucleic acids. Similarly, thenucleic acid encoding the growth factors can include additional codingsequences that confer functional attributes on the encoded polypeptide.Such sequences include secretion signal sequences (as shown in FIG. 1Aand SEQ ID NO:3).

Nucleic acids encoding growth factors that enhance bone growth can bemanipulated with standard procedures such as restriction enzymedigestion, fill-in with DNA polymerase, deletion by exonuclease,extension by terminal deoxynucleotide transferase, ligation of syntheticor cloned DNA sequences, site-directed sequence-alteration viasingle-stranded bacteriophage intermediate or with the use of specificoligonucleotides in combination with PCR or other in vitroamplification. These procedures are well known to those of ordinaryskill in the art, and exemplary protocols can be found, e.g., inSambrook and Ausubel (supra).

A polynucleotide sequence (or portions derived from it) such as a cDNAencoding an osteogenic growth factor can be introduced into a vector,such as a eukaryotic expression vector, by conventional techniques. Anexpression vector is designed to permit the transcription of thepolynucleotide sequence encoding the growth factor in cells by providingregulatory sequences that initiate and enhance the transcription of thecDNA and ensure its proper splicing and polyadenylation. Numerousexpression vectors are known to those of skill in the art, and areavailable commercially, or can be assembled from individual componentsaccording to conventional molecular biology procedures, such as thosedescribed in, e.g., Sambrook and Ausubel, cited above. The pY vectors(MLV-based vectors) described in the Examples is one such suitableexpression vector. Numerous other suitable vectors can be selected bythose of ordinary skill in the art.

The location of expression of the heterologus nucleic acid, e.g., FGF-2or an analog thereof, or another bone promoting factor, is dependent (atleast in part) on the promoter used to regulate expression of theencoded product. When a non-tissue specific promoter is used, theexpression occurs predominantly in the red marrow-rich regions, and atlow levels in circulating blood. Similarly, when an erythroid (e.g.,erythroblast) specific promoter is employed, expression of the nucleicacid is concentrated in the red marrow areas. In contrast, when anosteoblast specific promoter is employed, expression is restricted tothe areas enriched with osteoblasts and/or their precursors. In specificembodiments, the promoters used to direct expression of a polynucleotidethat encodes an osteogenic growth factor is an erythroid specificpromoter that effectively directes expression in hematopoietic stemcells, such as the ankyrin-1 promoter, an α-spectrin promoter, aζ-globin enhancer and/or sequences derived from the β-globin locuscontrol region (LCR).

For example, the cytomegalovirus (“CMV”) immediate early promoter is astrong constitutive promoter, which can be utilized to controltranscription of an osteogenic growth factor upon introduction of anexpression vector containing a nucleic acid encoding the osteogenicgrowth factor operably linked to the CMV promoter. Additionally, vectorscontaining the promoter and enhancer regions of the SV40 or longterminal repeat (LTR) of the Rous Sarcoma virus and polyadenylation andsplicing signal from SV40 are readily available (Mulligan et al., Proc.Natl. Acad. Sci. USA 78:1078-2076, 1981; Gorman et al., Proc. Natl.Acad. Sci. USA 78:6777-6781, 1982). Alternatively, the level ofexpression of the polynucleotide that encodes the growth factor can bemanipulated by using promoters that have different activities (forexample, the baculovirus pAC373 can express cDNAs at high levels in S.frugiperda cells (Summers and Smith, In Genetically Altered Viruses andthe Environment, Fields et al. (Eds.) 22:319-328, 1985, CSHL Press, ColdSpring Harbor, N.Y.).

Optionally, an inducible or otherwise regulatable promoter is utilized.For example, when the goal is fracture repair, the therapeutic gene isintroduced into the host cells operably linked to a transcriptioncontrol sequence that can be “turned on” upon administration of aninducing agent. One exemplary inducible promoter includes a Tetracyclineinducible element (e.g., a Tet/on transcription control sequence).Following introduction of the cells, tetracycline is administered, whichresults in the induction of expression. Once the therapeutic goal isobtained, e.g., fracture repair, attainment of a desired bone mass, theadministration of the inducing agent stops (for example, the subjectceases to take tetracycline), and expression of the heterologous nucleicacid ceases. Optionally, where even tighter regulation of expression isdesired, a suicide gene can be introduced into the host cells along withthe therapeutic nucleic acid. Expression of the suicide gene can beinduced once the therapeutic goal is reached, and the host cells arethereby eliminated.

In addition, some vectors contain selectable markers such as the gpt(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981) orneo (Southern and Berg, J. Mol. Appl. Genet. 1:327-341, 1982) bacterialgenes. These selectable markers permit selection of transfected cellsthat exhibit stable, long-term expression of the vectors (and thereforethe cDNA). The vectors can be maintained in the cells as episomal,freely replicating entities by using regulatory elements of viruses suchas papilloma (Sarver et al., Mol. Cell. Biol. 1:486, 1981) orEpstein-Barr (Sugden et al., Mol. Cell. Biol. 5:410, 1985).Alternatively, the vector can be selected to be integrated into genomicDNA. In either case, the introduced nucleic acid can be expressed on acontinuous basis.

Vector systems suitable for the expression of polynucleotides encodingosteogenic growth factors include, in addition to the specific vectorsdescribed in the examples, the pUR series of vectors (Ruther andMuller-Hill, EMBO J. 2:1791, 1983), pEX1-3 (Stanley and Luzio, EMBO J.3:1429, 1984) and pMR100 (Gray et al., Proc. Natl. Acad. Sci. USA79:6598, 1982). Vectors suitable for the production of intact nativeproteins include pKC30 (Shimatake and Rosenberg, Nature 292:128, 1981),pKK177-3 (Amann and Brosius, Gene 40:183, 1985) and pET-3 (Studiar andMoffatt, J. Mol. Biol. 189:113, 1986). The present disclosure, thus,encompasses recombinant vectors that comprise all or part of thepolynucleotides encoding osteogenic growth factors, for expression in asuitable host, either alone or as a labeled or otherwise detectableprotein.

Osteogenic Growth Factors

Another aspect of the disclosure relates to osteogenic growth factorsand methods for delivering them to the bone marrow of a subject toincrease bone formation. The disclosed osteogenic growth factors promotestem cell self-renewal and enhance bone formation in vivo. Optionally,such growth factors promote angiogenesis in vivo. Exemplary osteogenicgrowth factors include members of the FGF family of growth factors. Forexample, FGF family members that bind to and act via FGF-receptors 1, 2and/or 3, such as FGF-1, FGF-2, FGF-4, and analogs thereof, are used. Inother embodiments, the osteogenic growth factors are members of the Wntfamily of growth factors (such as Wnts 1, 2, 2B, 3, 3B, 4, 5A, 5B, 6,7A, 7B, 8A, 8B, 9A, 10A, 10B, 11 and 16). In alternative embodiments,the osteogenic growth factor is selected from among growth hormone (suchas human growth hormone), angiopoietins 1-7 (e.g., Angptl2, Angptl3),glial cell nerve factor, stem cell factor, parathyroid hormone (PTH),insulin like growth factor (IGF), platelet derived growth factor (PDGF),vascular endothelial growth factor (VEGF), Cox-2, and TGF-β. Thesegrowth factors are well known in the art, and their amino acid sequencesare readily accessible from publicly available databases, such asGENBANK®. One of skill in the art can easily obtain the amino acid of(and polynucleotide sequence that encodes) numerous such osteogenicgrowth factors by searching using the name (e.g., FGF-1 or fibroblastgrowth factor 1, etc.), or by entering all or a portion of an amino acidor polynucleotide sequence encoding the factor of interest. In additionto the wild-type or naturally occurring versions of the molecules listedabove, substantially similar polypeptides are also osteogenic growthfactors.

Substantially similar polypeptides can include variant polypeptides thatshare a substantial percentage of sequence similarity and that retainone or more functions of the reference polypeptide. Variants that retainsome or all of the functional attributes of the reference osteogenicgrowth factor are generally referred to as analogs. In some cases ananalog of an osteogenic growth factor has increased activity in vitro,ex vivo, and/or in vivo as compared to the naturally occurring growthfactor. Variants that retain some or all of the function of a referencegrowth factor typically possess no more than a small number of aminoacid substitutions (for example, 1, 2, 3, 4, 5, or 10 amino acidsubstitutions). The variant polypeptides typically have no more than 1%or 2% or 3% or 4% or 5%, or no more than about 10% amino aciddifferences with respect to the reference growth factor polypeptide.That is, the variant or analog polypeptide is at least about 90%, andtypically at least about 95%, or 96%, or 97%, or 98%, or even 99%identical to the reference growth factor. Accordingly, an osteogenicgrowth factor and related variants or analogs are typically encoded bypolynucleotide sequences with a high degree of sequence identity.Nonetheless, substantial divergence in the polynucleotide sequence canoccur due to the degeneracy of the genetic code without losing identitybetween the encoded products. More importantly, with respect to theencoded protein, even where an amino acid substitution is introduced,the mutation can be “conservative” and have no material impact on theessential functions of a protein. See Stryer, Biochemistry 3rd Ed.,1988.

Modifications of a polypeptide that involve the substitution of one ormore amino acids for amino acids having similar biochemical propertiesthat do not result in change or loss of a biological or biochemicalfunction of the polypeptide are designated “conservative” substitutions.These conservative substitutions are likely to have minimal impact onthe activity of the resultant protein. Table 1 shows amino acids thatcan be substituted for an original amino acid in a protein, and whichare regarded as conservative substitutions.

TABLE 1 Blosum Matrix of conservative amino acid substitutions. AminoAcid Conservative Substitutions G A, S, N P E D S, K, Q, H, N, E E P, D,S, R, K, Q, H. N N G, D, E, T, S, R, K, Q, H H D, E, N, M, R, Q Q D, E,N, H, M, S, R, K K D, E, N, Q, R R E, N, H, Q, K S G, D, E, N, Q, A, T TN, S, V, A A G, S, T, V M H, Q, Y, F, L, I, V V T, A, M, F, L, I I M, V,Y, F, L L M, V, I, Y, F F M, V, I, L, W, Y Y H, M, I, L, F, W W F, Y CNone

For example, one exemplary osteogenic growth factor is FGF-2 (SEQ IDNO:2). An exemplary polynucleotide sequence that encodes FGF-2 is shownin SEQ ID NO:1). One or more amino acid changes, or up to ten amino acidchanges (e.g., two substituted amino acids, three substituted aminoacids, four substituted amino acids, or five substituted amino acids,etc.) can be made in the polypeptide without losing function of theosteogenic growth factor.

For example, to improve stability of the osteogenic growth factor,FGF-2, an analog is produced that has an amino acid substituted for oneor more of the cysteines of wild type FGF-2. One cysteine can besubstituted with another amino acid residue. Alternatively, twocysteines can be substituted with other amino acid residues. If desired,three or even four of the cysteines can be substituted with other aminoacids. For example, in certain embodiments, a cysteine at position 70 issubstituted by another amino acid, or a cysteine at position 88 issubstituted by another amino acid, or cysteines at both position 70 andposition 88 are substituted by other amino acids. For example, stabilitycan be increased by substituting an amino acid that capable ofglycosylation (either by N-linked or O-linked glycosylation). Onespecific analog of FGF-2 is depicted in SEQ ID NO:4 (encoded by thepolynucleotide sequence of SEQ ID NO:3, or a polynucleotide sequencethat differs from SEQ ID NO:3 solely due to the degeneracy of thegenetic code). To enhance secretion of the FGF-2 analog, a secretionsignal sequence (BMP2/4 hybrid secretion signal) was added to thevariant FGF-2. Similar amino acid substituted analogs can be made forany osteogenic growth factor, and the function confirmed without undueexperimentation (e.g., by expressing a transgene encoding the growthfactor or analog in transplanted cells using the mouse bone marrowtransplantation model disclosed herein.

More substantial changes in a biochemical function or other proteinfeatures can be obtained by selecting amino acid substitutions that areless conservative than those listed in Table 1. Such changes include,for example, changing residues that differ more significantly in theireffect on maintaining polypeptide backbone structure (e.g., sheet orhelical conformation) near the substitution, charge or hydrophobicity ofthe molecule at the target site, or bulk of a specific side chain. Thefollowing substitutions are generally expected to produce the greatestchanges in protein properties: (a) a hydrophilic residue (e.g., seryl orthreonyl) is substituted for (or by) a hydrophobic residue (e.g.,leucyl, isoleucyl, phenylalanyl, valyl or alanyl); (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain (e.g., lysyl, arginyl, or histadyl)is substituted for (or by) an electronegative residue (e.g., glutamyl oraspartyl); or (d) a residue having a bulky side chain (e.g.,phenylalanine) is substituted for (or by) one lacking a side chain(e.g., glycine).

Additionally, part of a polypeptide chain can be deleted withoutimpairing or eliminating all of its functions. Similarly, insertions oradditions can be made in the polypeptide chain, for example, addingepitope tags, without impairing or eliminating its functions (Ausubel etal. (1997) J. Immunol. 159:2502). Other modifications that can be madewithout materially impairing one or more functions of a polypeptideinclude, for example, in vivo or in vitro chemical and biochemicalmodifications that incorporate unusual amino acids. Such modificationsinclude, for example, acetylation, carboxylation, phosphorylation,glycosylation, labeling, e.g., with radionuclides, and various enzymaticmodifications, as will be readily appreciated by those of ordinary skillin the art. A variety of methods for labeling polypeptides and labelsuseful for such purposes are well known in the art, and includeradioactive isotopes such as ³²P, fluorophores, chemiluminescent agents,enzymes, and antiligands.

Therapeutic Methods

The compositions and methods described herein can be used to expressosteogenic growth factors (such as FGF-2 or an analog thereof) inhematopoietic stem cells and/or hematopoietic progenitor cells fortherapeutic or prophylactic treatment of a condition or diseaseaffecting bone metabolism, for example bone growth (e.g., boneformation), bone maintenance and/or bone repair. For example, vectorsfor administering expressible nucleic acids with a beneficial (forexample, therapeutic) effect on bone tissue can be generated byincorporating a polynucleotide sequence that encodes an osteogenicgrowth factor operably linked to a constitutive, tissue specific orinducible promoter. When introduced into hematopoietic stem cells, whichare capable of homing to and engrafting the bone marrow, suchexpressible nucleic acids are expressed in skeletal tissue, reducingsafety concerns and increasing efficacy of delivery. Such applicationscan be used in the treatment of a condition or disease characterized byimpaired bone formation, e.g., due to aging, hormonal status, or geneticor physiological disorders, e.g., osteoporosis, osteogenesis imperfecta.This therapy utilizing osteogenic growth factors can also be used toenhance wound healing, especially the healing of bone fractures.

The nucleic acid encoding the selected osteogenic growth factor can beintroduced into a hematopoietic stem cell or progenitor cells in theform of a naked linear double stranded DNA. The polynucleotide sequenceencoding the osteogenic growth factor can be incorporated into a vector.Numerous suitable vectors are known in the art, and include, forexample, plasmids, viral vectors, and artificial chromosomes. Forexample, viral vectors are commonly used for in vivo or ex vivotargeting and therapy procedures are retroviral and DNA-based vectors.Methods for constructing and using viral vectors are known in the art(See e.g., Miller and Rosman, BioTech., 7:980-990, 1992). Preferably,the viral vectors are replication defective, that is, they are unable toreplicate autonomously in the target cell. In general, the genome of thereplication defective viral vectors that are used within the scope ofthe present disclosure lack at least one region that is necessary forthe replication of the virus in the infected cell. These regions caneither be eliminated (in whole or in part), or be renderednon-functional by any technique known to a person skilled in the art.These techniques include the total removal, substitution (by othersequences, in particular by the inserted nucleic acid), partial deletionor addition of one or more bases to an essential (for replication)region. Such techniques can be performed in vitro (i.e, on the isolatedDNA).

In some cases, the replication defective virus retains the sequences ofits genome that are necessary for encapsidating the viral particles. DNAviral vectors commonly include an attenuated or defective DNA viruses,including, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), Moloney leukemia virus (MLV) and human immunodeficiencyvirus (HIV) and the like. Defective viruses, that entirely or almostentirely lack viral genes, are preferred, as defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Thus, a specifictissue can be specifically targeted. Examples of particular vectorsinclude, but are not limited to, a defective herpes virus 1 (HSV1)vector (Kaplitt et al. Mol. Cell. Neurosci., 2:320-330, 1991), defectiveherpes virus vector lacking a glycoprotein L gene (See e.g., PatentPublication RD 371005 A), or other defective herpes virus vectors (Seee.g., WO 94/21807; and WO 92/05263); an attenuated adenovirus vector,such as the vector described by Stratford-Perricaudet et al. (J. Clin.Invest., 90:626-630 1992; La Salle et al., Science 259:988-990, 1993);and a defective adeno-associated virus vector (Samulski et al., J.Virol., 61:3096-3101, 1987; Samulski et al., J. Virol., 63:3822-3828,1989; and Lebkowski et al., Mol. Cell. Biol., 8:3988-3996, 1988).

For example, the gene can be introduced in a retroviral vector (e.g., asdescribed in U.S. Pat. Nos. 5,399,346, 4,650,764, 4,980,289 and5,124,263; all of which are herein incorporated by reference; Mann etal., Cell 33:153, 1983; Markowitz et al., J. Virol., 62:1120, 1988;PCT/US95/14575; EP 453242; EP178220; Bernstein et al. Genet. Eng.,7:235, 1985; McCormick, BioTechnol., 3:689, 1985; WO 95/07358; and Kuoet al. Blood 82:845, 1993). Most retroviruses are integrating virusesthat infect dividing cells. The Lentiviruses are integrating virusesthat infect nondividing cells. The retrovirus genome includes two LTRs,an encapsidation sequence and three coding regions (gag, pol and env).In recombinant retroviral vectors, the gag, pol and env genes aregenerally deleted, in whole or in part, and replaced with a heterologousnucleic acid sequence of interest. The gag, pol and env genes arecoexpressed in the packaging cell line. These vectors can be constructedfrom different types of retrovirus, such as, HIV, MoMuLV (“murineMoloney leukemia virus” MSV (“murine Moloney sarcoma virus”); RSV (“Roussarcoma virus”). In general, in order to construct recombinantretroviruses containing a nucleic acid sequence, a plasmid isconstructed that contains the LTRs, the encapsidation sequence and theconstruct of the present disclosure comprising a nuclear targetingsignal and a coding sequence. This construct is used to transfect apackaging cell line, which cell line is able to supply in trans theretroviral functions that are deficient in the plasmid. In general, thepackaging cell lines are thus able to express the gag, pol and envgenes. Such packaging cell lines have been described in the prior art,in particular the cell line PA317 (U.S. Pat. No. 4,861,719, hereinincorporated by reference), the PsiCRIP cell line (See, WO90/02806), andthe GP+envAm-12 cell line (See, WO89/07150). In addition, therecombinant retroviral vectors can contain modifications within the LTRsfor suppressing transcriptional activity as well as extensiveencapsidation sequences that can include a part of the gag gene (Benderet al., J. Virol., 61:1639, 1987). Recombinant retroviral vectors arepurified by standard techniques known to those having ordinary skill inthe art.

In one embodiment, the vector is an adenovirus vector. Adenoviruses areeukaryotic DNA viruses that can be modified to efficiently deliver anucleic acid of the disclosure to a variety of cell types. Variousserotypes of adenovirus exist. Of these serotypes, preference is given,within the scope of the present disclosure, to type 2 or type 5 humanadenoviruses (Ad 2 or Ad 5), or adenoviruses of animal origin (See e.g.,WO94/26914). Those adenoviruses of animal origin that can be used withinthe scope of the present disclosure include adenoviruses of canine,bovine, murine (e.g., May 1, Beard et al. Virol., 75-81, 1990), ovine,porcine, avian, and simian (e.g., SAV) origin. In some embodiments, theadenovirus of animal origin is a canine adenovirus, such as a CAV2adenovirus (e.g. Manhattan or A26/61 strain (ATCC VR-800)).

The replication defective adenoviral vectors described herein includethe ITRs, an encapsidation sequence and the nucleic acid of interest. Insome embodiments, at least the E1 region of the adenoviral vector isnon-functional. The deletion in the E1 region preferably extends fromnucleotides 455 to 3329 in the sequence of the Ad5 adenovirus(PvuII-BglII fragment) or 382 to 3446 (HinfII-Sau3A fragment). Otherregions can also be modified, in particular the E3 region (e.g.,WO95/02697), the E2 region (e.g., WO94/28938), the E4 region (e.g.,WO94/28152, WO94/12649 and WO95/02697), or in any of the late genesL1-L5.

In other embodiments, the adenoviral vector has a deletion in the E1region (Ad 1.0). Examples of E1-deleted adenoviruses are disclosed in EP185,573, the contents of which are incorporated herein by reference. Inanother embodiment, the adenoviral vector has a deletion in the E1 andE4 regions (Ad 3.0). Examples of E1/E4-deleted adenoviruses aredisclosed in WO95/02697 and WO96/22378. In still another embodiment, theadenoviral vector has a deletion in the E1 region into which the E4region and the nucleic acid sequence are inserted.

The replication defective recombinant adenoviruses according to thisdisclosure can be prepared by any technique known to the person skilledin the art (See e.g., Levrero et al. Gene 101:195, 1991; EP 185 573; andGraham EMBO J., 3:2917, 1984). In particular, they can be prepared byhomologous recombination between an adenovirus and a plasmid, whichincludes, inter alia, the DNA sequence of interest. The homologousrecombination is accomplished following co-transfection of theadenovirus and plasmid into an appropriate cell line. The cell line thatis employed should preferably (i) be transformable by the elements to beused, and (ii) contain the sequences that are able to complement thepart of the genome of the replication defective adenovirus, preferablyin integrated form in order to avoid the risks of recombination.Examples of cell lines that can be used are the human embryonic kidneycell line 293 (Graham et al. J. Gen. Virol. 36:59, 1977), which containsthe left-hand portion of the genome of an Ad5 adenovirus (12%)integrated into its genome, and cell lines that are able to complementthe E1 and E4 functions, as described in applications WO94/26914 andWO95/02697. Recombinant adenoviruses are recovered and purified usingstandard molecular biological techniques that are well known to one ofordinary skill in the art.

For in vivo administration, an appropriate immunosuppressive treatmentcan be employed in conjunction with the viral vector (e.g., adenovirusvector), to avoid immuno-deactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin-12 (IL-12), interferon-gamma (IFN-γ), or anti-CD4 antibody,can be administered to block humoral or cellular immune responses to theviral vectors. In addition, it is advantageous to employ a viral vectorthat is engineered to express a minimal number of antigens.

The adeno-associated viruses (AAV) are DNA viruses of relatively smallsize that can integrate, in a stable and site-specific manner, into thegenome of the cells that they infect. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies. The AAV genome has been cloned, sequenced andcharacterized. It encompasses approximately 4700 bases and contains aninverted terminal repeat (ITR) region of approximately 145 bases at eachend, which serves as an origin of replication for the virus. Theremainder of the genome is divided into two essential regions that carrythe encapsidation functions: the left-hand part of the genome, thatcontains the rep gene involved in viral replication and expression ofthe viral genes; and the right-hand part of the genome, that containsthe cap gene encoding the capsid proteins of the virus.

The use of vectors derived from the AAVs for transferring genes in vitroand in vivo has been described (See e.g., WO 91/18088; WO 93/09239; U.S.Pat. Nos. 4,797,368; 5,139,941; and EP 488 528, all of which are hereinincorporated by reference). These publications describe variousAAV-derived constructs in which the rep and/or cap genes are deleted andreplaced by a gene of interest, and the use of these constructs fortransferring the gene of interest in vitro (into cultured cells) or invivo (directly into an organism). The replication defective recombinantAAVs can be prepared by co-transfecting a plasmid containing the nucleicacid sequence of interest flanked by two AAV inverted terminal repeat(ITR) regions, and a plasmid carrying the AAV encapsidation genes (repand cap genes), into a cell line that is infected with a human helpervirus (for example an adenovirus). The AAV recombinants that areproduced are then purified by standard techniques.

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Methods for formulating and administering naked DNA tomammalian muscle tissue are disclosed in U.S. Pat. Nos. 5,580,859 and5,589,466. Other molecules are also useful for facilitating transfectionof a nucleic acid in vivo, such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g.,WO96/25508), or a cationic polymer (e.g., WO95/21931). Thus, the nucleicacids that encode osteogenic growth factors can be introduced into cellsin the form of naked or complexed DNA according to the teachings ofthese references, which are incorporated herein by reference.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker (Felgneret. al. Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987; Mackey, et al.Proc. Natl. Acad. Sci. USA 85:8027-8031, 1988; Ulmer et al. Science259:1745-1748, 1993). The use of cationic lipids can promoteencapsulation of negatively charged nucleic acids, and also promotefusion with negatively charged cell membranes (Felgner and RingoldScience 337:387-388, 1989). Particularly useful lipid compounds andcompositions for transfer of nucleic acids are described in WO95/18863and WO96/17823, and in U.S. Pat. No. 5,459,127, herein incorporated byreference.

DNA vectors can be introduced into the desired host cells by methodsknown in the art, including but not limited to transfection,electroporation (for example, into cells ex vivo or in vivo usingtranscutaneous electroporation), microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a genegun, or use of a DNA vector transporter (See e.g., Wu et al. J. Biol.Chem., 267:963-967, 1992; Wu and Wu J. Biol. Chem., 263:14621-14624,1988; and Williams et al. Proc. Natl. Acad. Sci. USA 88:2726-2730,1991). Receptor-mediated DNA delivery approaches can also be used(Curiel et al. Hum. Gene Ther., 3:147-154, 1992; and Wu and Wu, J. Biol.Chem., 262:4429-4432, 1987).

According to the methods disclosed herein, a nucleic acid encoding anosteogenic growth factor is introduced into a hematopoietic stem cell ora hematopoietic progenitor cell that is capable of homing to andengrafting the bone marrow following introduction into a subject. Thehematopoietic stem cells selected as donor cells can be derived fromsources other than the subject to be treated, such as a histocompatibledonor. In order to maximize the likelihood of stable bone marrowengraftment and survival of the transplanted cells, it is generallypreferable to obtain donor cells from the subject (that is, autologousdonor cells). Suitable donor cells can be obtained from the subject'sbone marrow or peripheral blood (including cord blood if available). Forexample, hematopoietic stem cells, such as CD34⁺ or Sca-1⁺ stem cells,can be isolated from bone marrow (or peripheral or cord blood) andenriched, e.g., using magnetic beads conjugated to an anti CD34 oranti-Sca-1 antibody. For example, human stem cells that are CD34⁺,AC133⁺, lin⁻, CD45⁻, CXCR4⁺ (Kucia et al., Leukemia 19:1118-1177, 2005)can be used in the context of the methods disclosed herein as vehiclesto express osteogenic growth factors encoded by heterologous nucleicacids. Importantly, such cells are capable of expressing therapeuticnucleic acids at appropriate levels, home to bone marrow throughout theentire skeleton, undergo stem cell renewal under appropriate conditions(e.g., exposure to FGF), and give rise to cells of the osteoblasticlineage. Additionally, hematopoietic stem cells characterized asCD34⁺/CD38⁺ cells (Chen et al., Stem Cells 15:368-377, 1997); CD73⁺,STRO-1⁺, CD105⁺, CD34⁻, CD45⁻, CD144⁻ cells (Tuli et al., Stem Cells21:681-693); and CD29⁺, CD63⁺, CD81⁺, CD122⁺, CD164⁺, cMet⁺, bonemorphogenetic protein receptor 1B⁺, and neurotrophic tyrosine kinasereceptor 3⁺ and CD34⁻, CD36⁻, CD45⁻, CD117⁻ (cKit⁻), and HLA-DR⁻(D'Ippolito et al., Journal of Cell Science 117:2971-2981, 2004) arealso human pluripotent hematopoietic stem cells. In certain embodiments,the hematopoietic stem cells express CXCR4, which facilitates homing tothe bone marrow. Additionally, the stem cells can express one or moreFGF receptors. Alternatively, CXCR4 expressing stem cells other thanhematopoietic stem cells, such as endothelial stem cells or mesenchymalstem cells can be used.

Such cells are suitable donor cells in the context of the methods andcompositions described herein. Alternatively, embryonic stem cells canbe employed as donor cells to increase bone growth and/or enhancehealing.

To increase the recovery of autologous hematopoietic stem cells, asubject can be treated with a growth factor that promotes expansion ofhematopoietic lineage cells, such as granulocyte colony stimulatingfactor (GCSF), granulocyte macrophage colony stimulating factor (GMCSF)or erythropoietin (EPO), or a combination of two or more of GCSF, GMCSFand EPO, to stimulate formation of red marrow throughout the skeleton.Hematopoietic stem cells and/or hematopoietic progenitors can then berecovered in increased numbers by standard procedures, such asapheresis. A nucleic acid encoding FGF-2 or an analog thereof, oranother factor that promotes increased bone growth is introduced intothe hematopoietic stem or progenitor cells as discussed above, and thecells are then introduced into the subject, e.g., injected or transfusedintravenously using standard procedures employed in the art for bonemarrow transplantation. Hematopoietic stem cells are capable ofachieving systemic engraftment even when relatively small numbers ofcells are introduced. For example, as few as 1000 to 10,000 transducedCD34⁺, CXCR4⁺ stem cells can be transplanted into a subject. Morecommonly, however, at least a million transduced CD34+ cells areintroduced into a recipient to effect systemic bone growth and/orenhance wound healing (e.g., of one or multiple fractures). Typically,between 1 and 10×10⁶ CD34⁺ stem cells are introduced into the subject.Following introduction into the subject, the cells have the ability toseek out (home to) and engraft the bone marrow. The host cells dividewithin the bone marrow to produce progeny cells, which express theintroduced gene, and its protein product, at the endosteal bone surfacesof the bone marrow. Increased expression of such factors induces boneformation, e.g., by stimulating and recruiting nearby stromal cells inthe marrow space to mature into osteoblasts.

Because bone growth (and, e.g., repair of bone fractures) is enhanced bymechanical loading of the bone, the methods described above can becombined with regimens involving or simulating physical loading of thebone. Such therapies increase bone growth at the desired locationswithin the skeletal system, and can be accomplished even in subjectsthat are immobilized or otherwise unable to undergo substantial physicalactivity. In addition to exercise, and/or physical therapy, treatmentmodalities employing vibration, e.g., ultrasound vibration, have beenfound to simulate mechanical loading and enhance bone growth.Additionally, because the methods described herein result in rapid andsubstantial bone growth, these therapies are most advantageouslycombined with administration of calcium and vitamin D at levelssufficient to prevent calcium deficiency that may otherwise occur withintense bone formation. For example, calcium intake can be coordinatedwith the degree or intensity of the formation to avoid calciumdeficiency. In addition, to optimize the effects of the osteogenicgrowth factor expressed by the hematopoietic stem cell and its progeny,in some cases additional growth factors (including for example,osteogenic growth factors and cytokines). Accordingly, the compositionsand methods for disclosed herein are suitable for treating subjects withbone wasting and/or bone fragility disorders, regardless of age, gender,or mobility status.

Bone Marrow Transplantation Model

Another aspect of the present disclosure relates to a novel bone marrowtransplantation model. Gene and cell therapies via bone marrowtransplantation (BMT) represents an exciting approach for treatingnumerous medical conditions, including, but not limited to, hereditaryimmunodeficiencies (Yang, Med. Sci. Monit. 10(7):RA155-165, 2004;Buckley, Annu. Rev. Immunol. 22:625-655, 2004; Fischer et al., Semin.Hematol. 41(4):272-278, 2004), hemoglobinopathies (Puthenveetil et al.,Curr. Hematol. Rep. 3(4):298-305, 2004), cancers (Fuchs, Curr. Opin.Mol. Ther. 6(1):48-53, 2004), and skeletal disorders (Klamut et al.,Crit. Rev. Eurkaryot. Gene Expr. 14(1-2):89-136, 2004). Prior toclinical trials of these applications in humans, appropriate animalmodels are used to further assess therapeutic efficacy and safetymonitoring. Accordingly, a number of BMT animal models have beendeveloped. The most widely studied animal model of BMT, e.g., for genetherapy, is the murine hematopoietic stem cells (HSCs) transplantationmodel.

Long-term engraftment of donor cells is essential for the success ofBMT-based approaches. BMT engraftment is affected by a number ofvariables, such as recipient preconditioning, HSC numbers and purity,route of delivery of donor cells, and ancillary treatments of HSCs.Accordingly, there have been a number of variations in strategy of thismodel addressing some of the variables to enhance engraftment. Eachvariation in strategy has its own advantages and disadvantages. Forexample, engraftment has been demonstrated in non-myeloablatedrecipients in some studies (Micklem et al., Transplantation6(2):299-302, 1968; Brecher et al., Blood Cells 5(2):237-246, 1979; Saxeet al., Exp. Hematol. 12(4):277-283, 1984; Stewart et al., Blood81(10):2566-2571, 1993). The advantage of this strategy is that itcaused low morbidity and mortality in the recipients; whereas a majordisadvantage is that it requires large cell doses (Ramshaw et al., Biol.Blood Marrow Transplant. 1(2):74-80, 1995) and/or infusions over severaldays (Stewart et al., Blood 81(10):2566-2571, 1993; Brecher et al.,Proc. Natl. Acad. Sci. USA 79(16):5085-5087, 1982). On the other hand,successful engraftment with cells numbers as low as a single HSC hasbeen documented in myeloablated recipients (Osawa et al., Science273(5272):242-245, 1996; Krause et al., Cell 105(3):369-377, 2001;Matsuzaki et al., Immunity 20(1):87-93, 2004), but this strategy exposesrecipients to lethal doses of irradiation (which leads to significanthost morbidity and mortality) and requires stringent maintenance ofaseptic conditions (which is needed to minimize the infection inresponse to myeloablation). Mild or sublethal irradiation falls betweenthese two extremes yielding engraftment with transplantation ofintermediate numbers of cells and reduced morbidity and mortality(Mardiney et al., Blood 87(10:4049-4056, 1996; Stewart et al., Blood91(10):3681-3687, 1998; James et al., Blood 96(4):1334-1341, 2000;Tomita et al., Blood 83(4):939-948, 1994). However, the engraftmentunder these conditions is highly inconsistent. An alternative approachto myeloablation is to use genetically myelosuppressed recipient mice.Accordingly, murine strains resulting from deletions or mutations in theW locus can be used as recipients. The W locus encodes the c-kit gene(Nocka et al., EMBO J. 9:1805-1813, 1990; Reith et al., Genes Dev.4:390-400, 1990). Mice with mutations in this gene are hematopoieticdeficient (Geissler et al., Genetics 97:337-361, 1981; Geissler et al.,Exp. Hematol. 11:452-460, 1983). The advantage of the w⁻/w⁻ recipientmice is avoidance of irradiation, but the disadvantage has been lowerlevels of engraftment than in preconditioned mice (Trevisan et al.,Blood 88:4149-4158, 1996; Soper et al., Exp. Hematol. 27:1691-1704,1999).

The present disclosure provides an improved murine HSC-basedtransplantation strategy that affords a consistent and high level oflong-term engraftment. This transplantation system provides a variety ofdesirable characteristics of previous strategies and at the same timeminimizes their limitations. This system incorporates the followingbeneficial features. HSC enriched Sca-1⁺ cells are used fortransplantation donor cells because of their propensity of homing tobone (Plett et al., Blood 102:2285-2291, 2003). Donor mice are selectedto be genetically or phenotypically distinguishable from the recipientmice.

For example, donor mice can be distinguished from recipient mice by thepresence of a marker that is not present in the recipient. The markercan be a simple genetic difference, such as a nucleic acid polymorphismthat does not result in a phenotypic difference, for example, a singlenucleotide polymorphism (SNP) detectable by molecular analysis.Alternatively, the marker can yield a phenotypic difference, such as anoptically detectable difference. Examples of optically detectablemarkers include proteins, such as green fluorescent proteins that confera particular optical property (fluorescence) on a cell. Additionally,markers include proteins that indirectly confer such a property byenzymatically converting a substrate to a detectable product. ExemplaryGFPs suitable as markers in the context of this disclosure includewithout limitation GFPs and variants described by Chalfie et al.,Science, 263:802-805, 1994; Heim et al., Proc. Natl. Acad. Sci. USA,91:12501-12504, 1994; Heim et al., Nature, 373:663-664, 1995; Peelle etal., J. Protein Chem., 20:507-519, 2001; and Labas et al., Proc. Natl.Acad. Sci. USA, 99:4256-4261, 2002, and in U.S. Pat. Nos. 6,818,443;6,800,733; 6,780,975; 6,780,974; 6,723,537; 6,265,548; 6,232,107;5,976,796; and 5,804,387. Red fluorescent proteins are described in,e.g., U.S. Pat. No. 6,723,537. Such fluorescent proteins can beoptically detected using, for example, flow cytometry. Flow cytometryfor GFP is described in, e.g., Ropp et al., Cytometry, 21:309-317, 1995,and in U.S. Pat. No. 5,938,738. Other suitable detection methods includea variety of multiwell plate fluorescence detection devices, e.g., theCYTOFLUOR 4000® multiwell plate reader from Applied Biosciences. Othermarkers include proteins with enzymatic activities that convert afluorogenic or chromogenic substrate into a fluorescent or visibleproduct. Examples of such enzymatic markers include various naturallyoccurring and modified luciferases. Exemplary luciferases are describedin U.S. Pat. Nos. 6,552,179; 6,436,682; 6,132,983; 6,451,549; 5,843,746(biotinylated); U.S. Pat. No. 5,229,285 (thermostable), and U.S. Pat.No. 4,968,613. U.S. Pat. No. 5,976,796 describes a luciferase-GFPmarker. Additional examples of markers with enzymatic activity include,e.g., chloramphenicol acetyltransferase (CAT), β-glucuronidase,β-galactosidase and alkaline phosphatase. Markers also includeselectable markers, the activity of which can be detected as resistanceor sensitivity to a selection agent, such as an antibiotic. Exemplaryselectable markers include thymidine kinase, neomycin resistance,kanamycin resistance, and ampicillin resistance.

In one example, Sca-1⁺ cells, isolated from green fluorescent protein(GFP)-expressing transgenic mice, are used as donor cells to distinguishdonor cells from cells of host origin. The Sca-1⁺ donor cells areinjected into sublethally irradiated, genetically myelosuppressedW⁴¹/W⁴¹ recipient mice through retroorbital vein from 2 to 24 hourspost-irradiation. Sublethally irradiated (e.g., 50-1000 cGy, such asabout 500 cGy) W⁴¹/W⁴¹ recipient mice injected with Sca-1⁺ cellstransduced with a lentiviral vector expressing GFP showed long-termengraftment and GFP transgene expression in every recipient mouse. Thus,this system provides an improved murine HSC-based transplantationstrategy that leads to consistent, robust, long-term engraftment withreduced morbidity and/or mortality.

Screening Methods

The mouse transplantation system described above provides a usefulsystem for drug screening (e.g., in cell culture and animal models). Thecellular and animal models are useful in methods for identifying agentsthat ameliorate the condition and/or that exert a favorable effect onbone metabolism, e.g., bone growth, bone maintenance, fracture repair,etc. In addition, such models are useful for evaluating potential genetherapies prior to their evaluation in human subjects.

For example, the methods of the present disclosure can be used togenerate cells to evaluate expression of a transgene of interest (e.g.,an osteogenic growth factor) and/or to determine whether administrationof a polypeptide or protein produced using recombinant technology (suchas an osteogenic growth factor) is effective for increasing bone growth.In addition, this transplantation model can be used to evaluatecompounds or compositions that are members of a library of potentialtherapeutic agents. Test compounds selected from the library areadministered to the cell or animal and the effect of the test compoundson bone growth, or an indicator associated with bone growth isevaluated. In the context of drug screening, indicators of bone growthinclude: a change in hematopoietic stem cell survival, a change inhematopoietic progenitor cell survival, a change in osteoblast lineagecell survival, a change in bone marrow engraftment by at least one of ahematopoietic stem cell, a hematopoietic progenitor cell, and anosteoblast lineage cell, a change in expression of one or more genes inat least one of a hematopoietic stem cell, a hematopoietic progenitorcell and an osteoblast lineage cell, a change in activity of one or moregene product in at least one of a hematopoietic stem cell, ahematopoietic progenitor cell, or an osteoblast lineage cell, a changein angiogenesis, a change in bone metabolism, a change in bone mass, anda change in bone density. Most typically, an increase in one or more ofthese indicators is measured or detected to identify and agent thatincreases bone growth and/or enhances bone repair.

The test compounds of the present disclosure can be obtained using anyof the numerous approaches in combinatorial library methods known in theart, including biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone, which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckemann et al., J.Med. Chem. 37: 2678-85, 1994); spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are preferred for use withpeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam, Anticancer Drug Des. 12:145, 1997).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90:6909, 1993; Erb et al., Proc. Nad. Acad. Sci. USA 91:11422,1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Cho et al.,Science 261:1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl.33.2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061, 1994;and Gallop et al., J. Med. Chem. 37:1233, 1994.

Libraries of compounds can be presented in solution (e.g., Houghten,Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84,1991), chips (Fodor, Nature 364:555-556, 1993), bacteria or spores (U.S.Pat. No. 5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci. USA89:1865-1869, 1992) or on phage (Scott and Smith, Science 249:386-390,1990; Devlin Science 249:404-406, 1990; Cwirla et al., Proc. Natl. Acad.Sci. USA 87:6378-6382, 1990; Felici, J. Mol. Biol. 222:301, 1991).

EXAMPLES

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.Each of the publications cited herein is incorporated by reference inits entirety.

Example 1 Cloning and Analysis of a Secretable FGF-2 Analog

Materials and Methods

Human FGF-2 Expression Plasmids.

The full-length human FGF-2 cDNA was cloned by PCR amplification usingpurified RNA of normal human skin fibroblasts as the template. Briefly,sense and antisense primers were designed to contain the entire openreading frame: sense primer: 5′-gcg cgc aag ctt G*TG GCA GCC GGG AGC ATCAC-3′ (SEQ ID NO:7); antisense primer: 5′-gcg get gac GGC CAT TAA AATCAG CTC TT-3′ (SEQ ID NO:8). HindIII and SalI restriction sites(underlined) were added to the sense and antisense primer, respectively,to facilitate its cloning into pFLAGCMV-1 cloning vector. The senseprimer contains the first 6 codons of the human FGF-2 gene with thestart codon for methionine (ATG) mutated to valine (GTG). This mutationwas introduced so that a secretion signal sequence could be added later.The antisense primer corresponds to the 3′-end of the open readingframe, including the termination codon. A single band of a 500-bp PCRproduct was obtained and its identity was confirmed by restrictionmappings. The PCR product containing the full length human FGF-2 cDNAwas then digested with HindIII/SalI, purified, and subcloned into thepFLAGCMV-1 cloning plasmid (Eastman Kodak, Rochester, N.Y.) to producethe pFC-FGF-2 plasmid vector. To enhance FGF-2 translation, an optimizedKozak sequence (gcccaccatgg) was later added to the full length humanFGF-2 cDNA. The 27 nucleotide sequence corresponding to 5′-UTR regionand a PstI restriction site were also added to the 5′ end to facilitatesubsequent cloning into the VR1012 cloning vector. This was alsoaccomplished by PCR amplification using the following sense primer:5′-aaa ctg cag GGG ATC CCG GCC GGG CCC CGC AGG ATG GCA GCC GGG AGC ATCAC-3′ (SEQ ID NO:9) and antisense primer: 5′-gcg get gac GGC CAT TAA AATCAG CTC TT-3′ (SEQ ID NO:10). The purified PCR product was digested withPstI and SalI restriction enzymes and subcloned into PstI/SalI-digestedVR1012 expression vector to generate the FGF-2 plasmid vector,VR1012-bFGF.

MLV-based FGF-2 expression vectors. To produce an MLV-based FGF-2vector, pY-FGF, the FGF-2 gene from VR1012-bFGF was excised by NotI andXhoI digestion, blunt-ended, and subcloned into BamHI-cleaved andblunt-ended MLV-based retroviral expression vector as describedpreviously (Peng et al., Mol. Therapy. 4:95-104; 2001). To add theBMP2/4 secretion signal sequence, the secretion signal sequence of theBMP2/4 hybrid gene (Hammonds et al., Mol. Endocrinol. 5:149-155, 1991)was subcloned into the 5′ end of the FGF-2 gene to produce thepY-BMP-FGF vector as described previously (Peng et al., Mol. Therapy.4:95-104; 2001). The exemplary FGF-2 analog is schematically illustratedin FIG. 1A. The start codon for methionine was removed during theinsertion of the BMP2/4 signal sequence. The amino acid sequence of theBMP-FGF junction area was shown in FIG. 1B.

PCR-Based Site-Directed Mutagenesis.

The QuickChange site-directed mutagenesis kit from Stratagene (La Jolla,Calif.) was used to generate mutations on cys-70 (C2) and/or cys-88 (C3)of FGF-2. Briefly, five pairs of complementary primers were synthesized:the first pair was used for the C2S mutation, the second pair for theC3S mutation, the third pair for the C3N mutation, and the fourth andfifth pair is for the C2A mutation and C3A mutation, respectively (Table2). The sequences of the primer sets are shown in Table 2. For C2Smutation, the cys-70 codon (TGT) was mutated to a serine codon (TCT).For the C3S mutation, the cys-88 codon (TGT) was mutated to a serinecodon (TCT). For the C3N mutation, the cys-88 codon was mutated to anasparagine codon (TCT). For C2A mutation, the cys-70 codon (TGT) wasmutated to an alanine (GCT). For the C3S mutation, the cys-88 codon(TGT) was mutated to a serine codon (GCT).

The PCR-based, site-directed mutagenesis procedure was carried outaccording to the recommendation of the vendor, using the pY-FGF orpY-BMPFGF parental plasmids as the template. For double mutations, themutation at the C2 and C3 was introduced sequentially. The resultingproducts were confirmed by DNA sequencing.

MLV-Based Vector Production.

The VSV-G pseudotyped MLV-based vectors were generated by transienttransfection in 293T cells as described previously (Peng et al., Mol.Therapy. 4:95-104; 2001). Briefly, a 10-cm plate of 293T cells weretransfected with a mixture of 20 μg of retroviral expression vector(e.g., pY-FGF or its derivatives), 10 μg of MLV-GP expression vector and1 μg of VSV-G expression vector by CaPO₄ precipitation. The conditionedmedium (CM) containing viral vectors was collected and concentrated byultracentrifugation 48 hrs after the transfection. The viral titer wasdetermined by the end-point dilution assay for the marker gene (e.g.,β-gal) expression or by real-time PCR assay for FGF-2 transgeneexpression in transduced HT1080 cells.

TABLE 2 Primers for PCR-based site-directed mutation of twocysteine residues of human FGF-2. Mutation Primer Sequence* C2S Sense5′-TCTATCAAAGGAGTGTCTGCTAACCGTTACCTG-3′ mutation Antisense SEQ ID NO: 115′-CGAGTAACGGTTAGCAGACACTCCTTTGATAGA-3′ SEQ ID NO: 12 C3S Sense5′-TTACTGGCTTCTAAATCTGTTACGGATGAGTGT-3′ mutation Antisense SEQ ID NO: 135′-ACACTCATCCGTAACAGATTTAGAAGCCAGTAA-3′ SEQ ID NO: 14 C3N Sense5′-TTACTGGCTTCTAAAAATGTTACGGATGAGTGT-3′ mutation Antisense SEQ ID NO: 155′-ACACTCATCCGTAACATTTTTAGAAGCCAGTAA-3′ SEQ ID NO: 16 C2A Sense5′-TCTATCAAAGGAGTGGCTGCTAACCGTTACCTG-3′ mutation Antisense SEQ ID NO: 175′-CGAGTAACGGTTAGCCGACACTCCTTTGATAGA-3′ SEQ ID NO: 18 C3A Sense5′-TTACTGGCTTCTAAAGCTGTTACGGATGAGTGT-3′ mutation Antisense SEQ ID NO: 195′-ACACTCATCCGTAACCGATTTAGAAGCCAGTAA-3′ SEQ ID NO: 20 *The underlinecodon indicates the site of the mutation.

Determination of the Amounts of FGF-2 Protein in Lysates or in CM.

Briefly, HT1080 cells or primary rat skin fibroblasts (RSF) in 6-wellplates were transduced with the test MLV-based vectors expressingwild-type or C2- and/or C3-mutated FGF-2 genes or BMP2/4-FGF-2 hybridgenes.

Following transduction, the cells were cultured in fresh medium prior tocollection of conditioned media (CM) and/or cell lysates. The CMs werecollected and frozen immediately until assay. The cell layers were lysedwith 500 μl of 1× Reporter lysis buffer (Promega, Madison, Wis.), andthe cell lysates were kept frozen until assay. The amount of FGFproteins in cell lysates and CMs was determined with an ELISA kit (R&Dsystems, Minneapolis, Minn.), according to the manufacturer'sinstruction. The amount of FGF-2 protein was normalized to ng of FGF-2per 10⁶ cells.

The identity of FGF-2 proteins in CMs and cell lysates was alsodetermined with Western immunoblot assays. Briefly, the CM and lysateproteins of HT1080 cells 24-hr after transduction with an MLV-basedvector expressing FGF-2 or an analog thereof were resolved on a 8% or15% SDS-PAGE and transblotted onto PVDF membranes (Bio-Rad Labs.,Hercules, Calif.). Known amounts of recombinant human FGF-2 standard(Sigma, St. Louis, Mo.) were included in each blot for comparison. Toidentify FGF-2 bands, the blots were incubated with a mouse anti-humanFGF-2 monoclonal antibody (UpState Biotech., Lake Placid, N.Y.) andFGF-2 bands were visualized with the goat-anti-mouse IgG antibodyconjugated with horse radish peroxidase (Pierce, Rockford, Ill.)followed by the chemiluminescent assay (Pierce).

Deglycosylation of CM FGF-2.

Deglycosylation was performed with enzymes provided by the Enzymaticdeglycosylation kit (Prozyme, San Leandro, Calif.) that contains PNGaseF (which hydrolyzes core oligosaccharides of N-linked glycosylatedproteins), endo-O-glycosidase (which hydrolyzes core oligosaccharides ofO-linked glycosylated proteins, only after substitutions on the coreoligosaccharide, such as sialic acid, galactose, fucose orN-acetylglucosamine, are removed with the appropriate exoglycosidase,such as sialidases), sialidase A (which removes sialic acid-containingsubstitutions from core oligosaccharides), and the combination of thethree glycosidases. Each glycosidase digestion reaction was carried outas recommended by the manufacturer. Briefly, the CMs were collected fromthe transduced cells, and 30 μl of each CM was mixed with 10 μl of 5×incubation buffer and 2.5 μl of denaturation solution. The samples wereheated to 100° C. for 5 minutes, followed by the addition of 2.5 μl ofdetergent solution. The PNGase, sialidase A, or endo-O-glycosidase (1 μleach), separately or all together, was added to the denatured CM proteinsamples. The samples were then incubated overnight at 37° C., and theFGF-2 proteins were then identified by Western analysis as describedabove.

Determination of Biological Activity of FGF-2 and Cysteine-MutatedVariants.

The in vitro biological activity of recombinant C2S/C3N-mutated FGF-2was determined by measuring its ability to stimulate [3H]thymidineincorporation in quiescent foreskin fibroblasts similar to a previouslydescribed assay (Kasperk et al., Growth Factors 3:147-158, 1990).Briefly, human foreskin fibroblasts were plated at a density of 5,000cells per well in 24-wells plates in serum-free DMEM for 24 hrs. Cellmedium was changed to fresh serum-free DMEM for an additional 24-hrs.Various amounts of the recombinant human FGF-2 protein standard and theCM FGF-2 protein of the pY-BMPFGFC2SC3N-transduced cells were then addedto each well of skin fibroblasts for an additional 24 hrs. The amount ofFGF-2 protein of each CM was pre-determined by an ELISA as describedabove prior to the bioactivity assay. [3H]Thymidine (1.5 μCi/well) wasadded during the final 6 hrs of the incubation. The incorporation of[3H]thymidine into trichloroacetic acid-precipitable DNA was measured inreplicate wells (n=6 per each group) by liquid scintillation counting.To confirm that the mitogenic activity was due to the FGF-2 protein inthe CM, the CM was also pretreated with 5 μg/ml of an anti-FGF-2antibody for an hour at 37° C. (to adsorb away the CM FGF-2 protein)prior to the mitogenic activity assay.

The in vivo biological activity of recombinant wild-type FGF-2 and therecombinant C2S/C3N-FGF-2 mutant was determined in a subcutaneous skinimplant rat model. Briefly, primary skin fibroblasts from inbred strainof Fisher 344 rats were transduced three times with each test MLVvector. The transduction level was determined with the β-gal marker geneexpression or FGF-2 expression and also by real-time PCR assay for FGF-2mRNA expression. Greater than 75% of transduction efficiency hasroutinely been obtained. Four million each of transduced primary ratskin fibroblasts (RSF) expressing β-gal marker gene, wild-type FGF-2gene, or C2S/C3N-FGF-2 mutant gene were incubated in a 1-cm×1-cm Gelfoamdisc (Pharmacia & Upjohn, Kalamazoo, Mich.) overnight as previouslydescribed (Gysin et al., Gene Therapy 9:991-999, 2002). Each disc wasimplanted into a subcutaneous pocket at the back of a Fisher 344 rat.Fourteen days later, the serum level of FGF-2 was determined with theELISA assay and the weight of each implant was determined.

Results

Effects of Cys-70 and/or Cys-88 Mutations on FGF-2 Secretion in HT1080Cells.

To evaluate whether C2 and/or C3 mutations improve FGF-2 proteinsecretion, HT1080 cells, which do not express detectable amounts ofFGF-2 protein, were transduced with MLV-based vectors expressingwild-type or the cysteine-mutated FGF-2 gene. Table 3 shows the amountsof FGF-2 protein in the cell lysate and CM of the transduced cells 48-hrpost-transduction.

TABLE 3 Effects of cys-70 (C2) and cys-80 (C3) mutations on FGF-2production and secretion in HT1080 cells (mean ± S.D., n = 2).^(a)Vector-treated cells FGF-2 in CM FGF-2 in lysate Total FGF-2^(b) %Secretion^(c) pY-FGF  16.2 ± 3.2^(d) 148.2 ± 39.5 164.4 ± 39.6   9.8 ±3.2 pY-FGFC2S 47.4 ± 9.3 497.6 ± 22.3 545.0 ± 24.2**  8.7 ± 1.8pY-FGFC3S  99.4 ± 15.8 532.2 ± 69.4 631.6 ± 71.2*  15.7 ± 3.2 pY-FGFC3N120.6 ± 4.0   503.6 ± 111.6 624.2 ± 111.7* 19.3 ± 4.3 pY-FGFC2SC3S 135.6± 9.0   843.2 ± 111.2 978.8 ± 111.6* 13.9 ± 2.1 pY-FGFC2SC3N 129.4 ±35.4 613.2 ± 2.6  742.6 ± 35.5** 17.4 ± 4.8 ^(a)There is no detectableFGF-2 protein in CM or lysate in untreated or pY-β-gal-treated HT-1080cells. ^(b)Total FGF-2 is the sum of FGF-2 in CM and FGF-2 in lysate. *p< 0.05, **p < 0.01, compared with the pY-FGF-treated cells. ^(c)% FGF-2secretion was calculated by dividing FGF-2 in CM by total FGF-2. None ofthe cysteine-mutated vector-treated cells was significantly differentfrom the pY-FGF-treated cells. ^(d)ng/10⁶ cells per 48 hrs.

Consistent with the premise that the secretion of unmodified FGF-2 inmammalian cells is inefficient, the amount of FGF-2 in the CM of thewild-type FGF-transduced cells (pY-FGF) represented ˜10% of the totalFGF-2 protein produced. Mutation of C2 or C3 to serine or asparaginealone significantly increased the amounts of FGF-2 in both cell lysatesand CMs by 2- to 3-fold. However, the FGF-2 secretion was enhanced by40-100% in cells transduced with vectors containing the C3 mutation (C3Sand C3N), but not those with the C2S mutation. Double mutations of C2and C3 increased the total amounts of FGF-2 about pY-FGF-transducedcells but did not further enhance FGF-2 secretion compared to the C3single mutation. Western immunoblot analysis showed that: a) each groupof transduced cells produced a major immunoreactive band of 21-kd, thatco-migrated with the FGF-2 protein standard, in both cell lysates andCMs, and b) the amounts of FGF-2 protein in both lysates and CMs weresignificantly higher in cells transduced with the cysteine-mutated FGF-2vectors than in cells transduced with the wild-type pY-FGF vector. Cellstransduced with the control pY-LMPHA vector, as expected, produced nodetectable amounts of FGF-2 protein in cell lysates or in CMs.

Effects of the Addition of BMP2/4 Secretion Signal Sequence and C2and/or C3 Mutation on FGF-2 Secretion in HT1080 Cells.

Table 4 shows that the addition of the BMP2/4 hybrid secretion signalsequence alone (pY-BMPFGF) resulted in a sevenfold reduction in totalamounts FGF-2 compared to the pY-FGF-transduced cells. However, therelative amount of FGF-2 protein in the CMs of the pY-BMPFGF-treatedcells accounted for 54.2% of the total FGF-2 proteins produced, whichwas >5-fold more than that in the CMs of the pY-FGF-treated cells(9.8%). This indicated that the BMP2/4 signal sequence significantlyenhanced the secretion of FGF-2 in HT1080 cells. The single C2S mutationalong with the BMP2/4 secretion signal sequence (pY-BMPFGFC2S) did notincrease total amount or secretion of the FGF-2 protein compared to thepY-BMPFGF-transduced cells. In contrast, double mutation of C2 and C3markedly and significantly increased total amount of FGF-2 protein inCMs and in lysates compared to the pY-BMPFGF group. The enhancement inthe total FGF-2 produced appeared to be larger in C3N mutated groupcompared to that in C3S mutated group: C3S mutation yielded a ˜3-foldincrease; while C3N mutation produced a 7-fold enhancement. The increasein the amounts of FGF-2 in CMs are also higher in cells transduced withvectors containing the C3N mutation than in cells treated with vectorscontaining the C3S mutation. With respect to FGF-2 secretion, the cellstreated with the C2/C3 double mutated vectors showed an increase inFGF-2 secretion by ˜40-fold compared with pY-BMPFGF-treated cells.

TABLE 4 Effects of the addition of BMP2/4 secretion signal sequence andC2/C3 mutations on FGF-2 production and secretion in HT1080 cells (mean± SD, n = 2). Vector-treated cells FGF-2 in CM FGF-2 in lysate^(d) TotalFGF-2^(a) % Secretion^(b) pY-FGF  16.2 ± 3.2^(c) 148.2 ± 39.5 164.4 ±39.6  9.8 ± 3.2  pY-BMPFGF 13.0 ± 1.0 11.0 ± 0.3 24.0 ± 1.0* 54.2 ±4.4** pY-BMPFGFC2S 15.6 ± 0.4 16.2 ± 3.1 31.8 ± 3.1* 49.1 ± 9.5* pY-BMPFGFC2SC3S 318.8 ± 5.1  153.2 ± 0.7  472.0 ± 5.1**  67.5 ± 1.1***pY-BMPFGFC2SC3N  916.2 ± 203.1 155.4 ± 31.8 1071.6 ± 205.6* 85.5 ± 25.8*^(a)Total FGF-2 is the sum of FGF-2 in CM and FGF-2 in lysate. *p <0.05, and **p < 0.01, compared with the pY-FGF-treated cells. ^(b)%FGF-2 secretion was calculated by dividing FGF-2 in CM by total FGF-2.*p < 0.05, **p < 0.01, and ***p < 0.001, compared with thepY-FGF-treated cells. ^(c)ng/10⁶ cells per 48 hrs. ^(d)ng/10⁶ cells.

Table 5 summarizes the effect of C2 and C3 mutations of FGF-2 productionin rat skin fibroblasts.

TABLE 5 Comparison of cys-70 (C2) and cys-80 (C3) mutations on FGF-2secretion in normal rat skin fibroblasts.^(a) Vector-treated cells FGF-2in CM (pg/10⁶ cells) pY-β-gal control 2.8 pY-FGF 15.8 pY-BMPFGF 35.3pY-BMPFGFC3A 3,641.6 pY-BMPFGFC2AC3A 2,586.5 pY-BMPFGFC2A 759.2pY-BMPFGFC2AC3N 18,674.2 pY-BMPFGFC2SC3S 3,255.2 pY-BMPFGFC2SC3N 8,480.6pY's denotes the MLV-based vectors; pY-BMP's means the BMP2/4 secretionsignal sequence has been added to the transgene to improve secretion;C2A = cys-70 to alanine mutation, C2S = cys-70 to serine mutation, C3A =cys-88 to alanine mutation; C3S = cys-88 to serine mutation; and C3N =cys-88 to asparagine mutation.

Rat skin fibroblasts secreted very low basal level of FGF-2 (2.8 pg/10⁶cells/24 hrs). Rat skin fibroblasts transduced with wild-type pY-FGFvector also secreted very low level of FGF-2 (15.8 pg/10⁶ cells/24 hrs).Addition of the BMP2/4 secretion signal only increased the amount ofFGF-2 in CM by ˜3-fold. Modification of cys-70 (C2) and cys-88 (C3)drastically increased FGF-2 secretion in rat fibroblasts. Modificationof cys-70 to alanine increased secretion at least 2-fold more than thecys-70 to serine mutation.

Western immunoblot of FGF-2 immunoreactive protein bands in lysates andCMs of HT1080 cells transduced with pY-BMPFGF vectors showed anadditional immunoreactive band of ˜26 kD, in addition to the 21-kDimmunoreactive protein band that co-migrated with the FGF-2 proteinstandard in lysates of cells transduced with the C2S vector and/or theC3S vector (but not the non-cysteine-mutated vectors). Lysates of cellstransduced with the C2SC3N vector showed yet another immunoreactive bandof ˜29 kD. Because CMs of cells transduced with vectors that did nothave the C3 mutation contained relatively low levels of FGF-2 protein(Table 4) and because only a small amount of CM (10 μl) was analyzed byWestern immunoblots, only CMs of cells transduced with vectorscontaining the C3 mutation showed detectable FGF-2 immunoreactive bands.Because serine and asparagine are potential sites of O- andN-glycosylation, respectively, it is possible that the largerimmunoreactive bands are glycosylated forms of FGF-2 and that the 26-kDband is O-glycosylated FGF-2 and the 29-kD band is both O- andN-glycosylated.

Effects of the Addition of BMP2/4 Signal Sequence and C2 and C3 DoubleMutations on FGF-2 Secretion in Rat Skin Fibroblasts.

The ability of the BMP2/4 signal sequence in combination with C2S andC3N double mutation to increase the stability and secretion of FGF-2protein in normal, untransformed cells was evaluated. Primary rat skinfibroblasts (RSF) were transduced with the pY-β-galactosidase (pY-β-gal)[control vector], the wild-type pY-FGF, or the cysteine-mutatedpY-BMP-FGF-C2SC3N vector. Staining for β-gal expression with x-gal inpY-β-gal-transduced cells indicated that the transduction efficiencywas >90%. Table 6 summarizes the amounts of FGF-2 protein in CM and inlysate of RSF 48-hr post-transduction. Both CM and lysates of RSFtransduced with pY-β-gal showed very low (almost undetectable) levels ofFGF-2 protein. Transduction of RSF with the pY-FGF vector markedlyincreased the FGF-2 protein level in the lysate, but the amount of FGF-2protein in CM was still very low, only accounting for ˜1% of the totalFGF-2 produced. As in HT1080 cells, the addition of the BMP2/4 secretionsignal inhibited FGF-2 biosynthesis in RSF, as the total FGF-2 proteinsynthesized by RSF transduced with the pY-BMPFGFC2SC3N vector werereduced by ˜60% compared to that the pY-FGF-treated cells. Thisreduction was not due to a reduced number of copies of inserted gene asdetermined by real-time PCR analysis on genomic DNA. The large majorityof the FGF-2 protein (˜90%) in the pY-BMPFGFC2SC3N-transduced cells wasin CMs, indicating that the inclusion of BMP2/4 signal sequence also ledto a drastic increase in FGF-2 secretion in untransformed, RSF. Moreimportantly, despite the ˜60% reduction in the total amounts of FGF-2produced, the actual amounts of FGF-2 in the CMs of thepY-BMPFGFC2SC3N-transduced RSF were 37-fold higher than those in CMs ofthe pY-FGF-transduced cells.

TABLE 6 Effects of the test modification of FGF-2 gene on FGF-2production and secretion in primary rat skin fibroblasts. Vector-treatedcells FGF-2 in CM FGF-2 in lysate^(d) Total FGF-2^(a) % Secretion^(b)pY-β-gal  0.00 ± 0.00^(c) 0.52 ± 0.02 0.52 ± 0.02 0.48 ± 0.09 pY-FGF0.88 ± 0.01 91.7 ± 0.0  92.6 ± 0.0  0.95 ± 0.10 pY-BMPFGFC2SC3N 33.0 ±0.4  4.8 ± 0.5 37.9 ± 0.6* 87.3 ± 1.7* ^(a)Total FGF-2 is the sum ofFGF-2 in CM and FGF-2 in lysate. *p < 0.05, and **p < 0.01, comparedwith the pY-FGF-treated cells. ^(b)% FGF-2 secretion was calculated bydividing FGF-2 in CM by total FGF-2. *p < 0.001, compared with thepY-FGF-treated cells. ^(c)ng/10⁶ cells per 48 hrs. ^(d)ng/10⁶ cells.

FGF-2 proteins in CMs of three preparations ofpY-BMPFGFC2SC3N-transduced RSF were compared to those in CM of apreparation of pY-BMPFGFC2SC3N-transduced HT1080 cells by westernimmunoblot. In contrast to HT1080 cells which produced the major 26-kDand 29-kD immunoreactive bands, the CM from each of the threepY-BMP-FGF-C2SC3N-transduced RSF preparations contained a major 27-kDimmunoreactive protein band and a very minor 29-kD band, in addition tothe 21-kD FGF-2 band. The production of this 27-kD band was not uniqueto primary RSF, since the transduced primary rat marrow stromal cellsalso yielded this major 27-kD band. Thus, the glycosylation of thecysteine-mutated FGF-2 proteins appears to be different between primarycells and transformed cells.

To confirm that the 26-kD and 29-kD immunoreactive bands in HT-1080cells and the 27-kD band in RSF were indeed glycosylated species ofFGF-2, the CM of pY-BMP-FGF-C2SC2N-treated HT1080 cells and also the CMof pY-BMP-FGF-C2SC2N-treated RSF were treated with PNGase F,endo-O-glycosidase, sialidase A, or all three glycosidases. With theHT1080 CM, combination treatment with all three glycosidases convertedboth the 26-kD and 29-kD bands to the 21-kD non-glycosylated FGF-2,indicating that both bands are glycosylated species of FGF-2. SialidaseA alone converted both the 26-kD and 29-kD bands to a band of anapparent size between 21-kD and 26-kD. The PNGase F treatment aloneconverted the 29-kD to a major band of slightly greater than 26-kD, inaddition to the 21-kD non-glycosylated FGF-2. The endo-O-glycosidasetreatment alone (without sialidase A) had no significant effects oneither the 29-kD or 26-kD band, suggesting that the O-linked coreoligosaccharide had sialic acid-containing substitutions. With respectto the 27-kD band in RSF CM, the combination treatment also convertedthe 27-kD band to 21-kD non-glycosylated FGF-2 band, confirming that itis a glycosylation form of FGF-2. However, the PNGase F treatment, butnot endo-O-glycosidase or sialidase A treatments, converted the 27-kDband to the 21-kD non-glycosylated FGF-2, suggesting that the 27-kD bandis primarily an N-linked glycosylated FGF-2.

Effects of Glycosylation on the Biological Activity of GlycosylatedFGF-2 Chimeric Protein.

The pY-BMPFGFC2SC3N-transduced RSF displayed an altered morphologycompared to pY-β-gal-transduced RSF. Primary rat skin fibroblasts weretransduced with either pY-β-gal control vector or the pY-BMPFGFC2SC3Nvector. One week after the transduction, the cells were stained withFast Red. The cells transduced with the pY-BMPFGFC2SC3N vector were muchsmaller than the pY-β-gal-transduced control cells. They were alsochanged to spindle-like shape from the elongated shape that is typicalof fibroblasts. This morphology was similar to that of primary RSF aftertreatment with FGF-2 for an extended period of time, e.g., 1 week. Nosuch morphological change was seen in the pY-FGF-transduced RSF. Thechange in cell morphology in pY-BMPFGFC2SC3N-treated RSF, which secretedlarge amounts of glycosylated FGF-2 into CMs, strongly suggests that thesecreted glycosylated FGF-2 is functionally active.

The ability of the modified FGF2* and the wild type, un-modified FGF2,to promote the proliferation of marrow stromal cells and to stimulatethe phosphorylation and activation of the extracelluar regulated proteinkinases-1/2 (Erk1/2) was also compared in vitro in the cellproliferation assay. There were no significant differences between themodified FGF2 and the unmodified WT FGF-2 in their ability to stimulatecell proliferation and Erk1/2 activation.

To confirm that the glycosylation did not adversely affect thebiological activity of FGF-2 in vitro, the ability of the CM of thepY-BMPFGFC2SC3N-treated cells to stimulate [³H]thymidine incorporationin quiescent human primary foreskin fibroblasts was compared with thatof the commercial, recombinant non-glycosylated FGF-2. Thedose-dependent stimulation curve of the CM FGF-2 of transduced HT1080cells was similar to that of the recombinant non-glycosylated FGF-2,demonstrating that glycosylation did not significantly affect thebiological activity of FGF-2. More importantly, the pretreatment of theCMs of pY-BMPFGFC2SC3N-treated cells with an anti-FGF-2 antibody almostcompletely eliminated the mitogenic activity of the CM, indicating thatthe mitogenic activity of the CM was due largely to FGF-2.

Biological Effects of Subcutaneous Implantation ofpY-BMPFGFC2SC3N-Transduced Rat Skin Fibroblasts in Syngenic Rats.

To initiate an evaluation of the utility of the pY-BMPFGFC2SC3N vectorin gene transfer protocols, subcutaneous implantation of RSF transducedwith the pY-BMPFGFC2SC3N vector into the dorsal back of syngenic ratswas evaluated, and the ability of such transplants to increase serumFGF-2 levels and enhance growth of the implant was determined. AGel-foam square (1 cm²) was impregnated with 4 million of primary ratskin fibroblasts transduced with pY-β-gal, pY-FGF control vector, orpY-BMPFGFC2SC3N vector overnight and was implanted subcutaneously intothe dorsal back of a syngenic rat. Fourteen days after the implantation,animals were sacrificed and serum FGF-2 was determined by an ELISA assay(FIG. 2). The implants were dissected, weighed and evaluatedmorphologically.

The serum FGF-2 level in rats with implants containing the controlpY-β-gal-transduced RSF or pY-FGF-transduced RSF were very low (<10ng/ml). By contrast, every rat that had the implant containing thepY-BMPFGFC2SC3N-transduced RSF showed very high serum FGF-2 levels (˜150ng/ml). The weight of implants was significantly greater (2- to 3-fold)in the pY-BMPFGFC2SC3N group than that in the pY-β-gal or in the pY-FGFcontrol groups at fourteen days. Gross anatomical examination of theimplants showed that the pY-BMPFGFC2SC3N implants, but not the pY-β-galor pY-FGF implants, were reddish in color and rich in blood,demonstrating that extensive vascularization had occurred.

The results described above demonstrate that efficient and consistentsecretion of FGF-2 from mammalian cells can be achieved by adding to theFGF-2 transgene a potent secretion signal sequence, such as the BMP2/4hybrid signal sequence, and also by mutation of cys-70 (C2) and cys-88(C3) to a serine or an asparagine. These modifications together have ledto an overall ˜60-fold and ˜40-fold increase in the actual amounts ofFGF-2 protein secreted into the CMs of transduced HT1080 cells andprimary RSF, respectively, compared to the unmodified FGF-2 gene. Moreimportantly, the ex vivo application of the modified FGF-2 MLV-basedexpression vector, but not the wild-type unmodified FGF-2 vector, in asubcutaneous transplant rat model resulted in a ˜200-fold increase inserum FGF-2. In addition, it also led to a ˜3-fold increase in thegrowth weight and the apparent vascularization of the implants. Thesefindings indicate that the secretion and stability can be greatlyenhanced by modifying wild type FGF-2 (for example, by the inclusion ofa strong secretion signal, such as the BMP2/4 secretion signal, and theby stabilizing the product, e.g., by the introduction of C2/C3mutations). Furthermore, these results show that a vector incorporatingmodified FGF-2 can be used to produce consistent, therapeutic levels ofFGF-2.

Because the C2/C3-mutated FGF-2 expression vectors have the same viralbackbone as the wild type unmodified FGF-2 expression vector, andbecause the viral titers and transduction efficiency (determined byreal-time PCR analysis of FGF-2 mRNA transcripts levels) of theC2/C3-mutated FGF-2 expression vectors in these studies were verysimilar to those of the unmodified FGF-2 vector, the increased amountsof FGF-2 proteins in cells transduced with the C2/C3-mutated vectorswere not due to an increase in gene expression and/or proteinbiosynthesis. Thus, the observed increase in total FGF-2 proteins incells transduced with the C2/C3-mutated FGF-2 vectors is due to anenhanced stability (and/or decreased degradation) of the recombinantFGF-2 protein. This conclusion is consistent with the previous studiesin COS-7 cells, which showed that the mutation of C2/C3 of human FGF-2gene leads to an increase in the stability of the recombinant FGF-2protein (Sasada et al., Ann. N.Y. Acad. Sci. 638:149-160, 1991; Seno etal., Biochem. Biophys. Res. Commun. 151:701-708, 1988). Without beingbound by theory, it is likely that, in addition to preventing theoxidation of key cysteines and/or intra- and/or inter-moleculardisulfide formation, which have been shown to adversely affect FGF-2stability (Iwane et al., Biochem. Biophys. Res. Commun. 146:470-477,1987), glycosylation of the introduced serine or asparagine residuesplays an important role in the stabilization of the recombinant FGF-2protein. This conclusion is supported by two observations. Firstly, theenhancement in FGF-2 stability in the CMs of HT1080 cells transducedwith C2/C3-mutated vectors without the BMP2/4 signal sequence was only˜7-fold, while the increase in FGF-2 stability in cells transduced withthe BMP2/4 signal sequence containing C2/C3-mutated vectors was˜60-fold. Secondly, the FGF-2 protein produced by HT1080 cellstransduced with the C2/C3-mutated vectors without the BMP2/4 secretionsignal sequence was largely non-glycosylated, whereas the FGF-2 proteinproduced by cells transduced with the C2/C3-mutated vectors with theBMP2/4 signal sequence was primarily glycosylated. In combination, theseresults indicate that glycosylation of the mutated FGF-2 protein playsan important role in enhancing production the FGF-2 recombinant protein.

The recombinant FGF-2 protein produced by cells transduced with thecysteine-mutated FGF-2 vectors were glycosylated only if BMP2/4secretion signal sequence was included in the construct. The BMP2/4secretion signal sequence is a member of the large family of cleavableclassical signal sequences (Hammonds et al., Mol. Endocrinol. 5:149-155,1991) that direct the secretory proteins to the ER and the golgi forprocessing, including glycosylation, and secretion (Brodsky, Int. Rev.Cytol. 178:277-328, 1998). Thus, the BMP2/4 signal sequence redirectedthe FGF-2 chimera to the ER/golgi for secretion. The glycosylation ofthe cysteine-mutated FGF-2 most likely took place within the ER/golgi,where glycosidases and glycosyltransferases are located (Verbert et al.,Biochim. Biophys. Acta 1473:137-146, 1999). These conclusions areconsistent with previous findings that the addition of the growthhormone secretion signal (another member of the cleavable classicalsignal sequences) to the FGF-2 gene also appeared to result in thesecretion of glycosylated FGF-2 proteins (Blam et al., Oncogene3:129-136, 1988).

Enhancement in FGF-2 stability was achieved by introducing a mutation atthe C3 site, in that the C3S mutation yielded a ˜3-fold enhancement, andthe C3N mutation produced a ˜6-fold increase. Although the mutation ofC2 and C3 (e.g., to an amino acid that can be glycosylated, such as aserine or an asparagine) appears to have an impact on the stability (ordegradation) of the FGF-2 recombinant protein, the C3 mutation has anenhancing effect (>2-fold) on the secretion of the recombinant FGF-2protein even without the BMP2/4 sequence signal.

The FGF-2 chimeric protein produced by cells transduced with themodified FGF-2 expression vector is biologically active. The transducedcells stimulated the proliferation of quiescent human skin fibroblaststo an extent similar to that induced by recombinant FGF-2 protein.Pre-incubation of the CMs with an anti-FGF-2 antibody completelyabolished the ability of the CMs to stimulate the proliferation ofquiescent skin fibroblasts. Hence, the mitogenic activity in the CMs ofthe transduced cells was due to the FGF-2 protein and not other growthfactors. In addition, the morphology of the cells transduced with themodified FGF-2, but not that of cells transduced with control vectors,was highly proliferative, as is expected with biologically active FGF-2.The ex vivo administration of cells transduced with the modified FGF-2vector also promoted the growth and vascularization of the implants toprovide further evidence that the FGF-2 protein produced by the modifiedexpression vector is biologically active in vivo.

Example 2 An Improved Mouse Sca-1⁺ Cell-Based Bone MarrowTransplantation Model

Materials and Methods

Animals.

The TgN β-actin-EGFP (TgN-GFP) donor mice, wild-type C57BL/6J mice andC57BL/6J-W⁴¹/W⁴¹ (W⁴¹/W⁴¹) mice were used to produce an improved mousebone marrow transplantation model.

Sca-1⁺ Cell Enrichment.

Whole bone marrow (WBM) cells were harvested from TgN-GFP mice byflushing tibiae and femurs with phosphate-buffered saline (PBS) using a26-g needle and syringe. Erythrocytes were removed by osmotic lysisusing a solution of 155 mM NH₄Cl, 10 mM KHCO₃ and 110 μM Na₂EDTA,followed by rinsing with PBS. The cell preparation was then incubatedwith magnetic microbeads conjugated with antibody specific for Sca-1 andapplied twice to an automated magnetic separation column (AutoMacs™)according to manufacturer instructions (Miltenyi Biotec, Inc, Auburn,Calif.). Cell yields of aliquots of the WBM, erythrocyte-lysed, andSca-1⁺ cell-enriched preparations were measured by manual cell count ofviable cells as determined by trypan dye exclusion. Recovery wascalculated by dividing the number of cells counted after the lysis oferthrocytes and Sca-1⁺ enrichment steps by the number of WBM cellsharvested. To assess enrichment efficiency, aliquots of each cellpreparation were incubated with either PE-conjugated Sca-1 specific orPE-conjugated rat isotype control antibody (Pharmingen, San Diego,Calif.) and analyzed for Sca-1 and/or GFP-expression with a FACSCaliburSystem (BD Biosciences, San Jose, Calif.). The percentage of Sca-1⁺cells was calculated by subtracting the value obtained with thePE-conjugated rat isotype control antibody from that obtained with thePE conjugated Sca-1 specific antibody.

Transplantation.

Two weeks before and 2 weeks after the irradiation procedure, recipientmice were provided sterile food and autoclaved, acidified water (pH2.0-2.5) containing 50 mg/L Neomycin SO₄ and 13 mg/L Polymixin B SO₄.Recipient mice were preconditioned by total body irradiation with usinga ⁶⁰Co source delivering a single radiation dose of 500 cGy at a rate of80 cGy per minute. For non-irradiated controls, sham irradiations wereperformed in parallel. Unless otherwise stated, Sca-1⁺ cells weretransplanted into recipients four hours or 24 hours after theirradiation via lateral tail vein or retroorbital injection. For thetail vein injection approach, 30 W⁴¹/W⁴¹ recipient mice weretransplanted via the tail vein injection method with 400,000 Sca-1⁺cells harvested from TgN-GFP donor mice. For the retroorbital injectionmethod, an aliquot of 400,000 Sca-1⁺ cells of the same donor cellpreparation was injected into each of the six W⁴¹/W⁴¹ recipient micewith via retroorbital plexus.

Analysis of Engraftment.

At various times post transplantation, peripheral blood was collectedvia the lateral tail vein. Erythrocytes were lyzed and FACS analysis wasperformed for GFP-expressing donor cells.

Preparation of eGFP Lentiviral Vector-Transduced Sca-1⁺ Cells forTransplantation.

Sca-1⁺ cells were isolated as described above and plated in 6-wellretronectin-coated plates at a density of 4×10⁶ cells/well in IMDM mediacontaining fetal bovine serum (FBS), human Flt-3 ligand, murine stemcell factor, murine IL-6, murine IL-1α and murine IL-3 and 100 μMadditional dNTPs. After 24 hours, unconcentrated MLV-based viral stockwas applied to the cells. After 8 hours the media was removed, and freshmedia with cytokines and viral stocks were reapplied. After anadditional 8 hours, the media was removed and fresh media with cytokines(without virus) were applied. Cells were then transplanted 12-24 hoursafter transduction.

Statistical Analysis.

Comparisons of differences between two variables were performed usingthe two-tailed, two-sample with equal variances, independent t-test.Comparison of multiple groups was performed with one-way ANOVA. Resultswere considered significant when p<0.05. All data are reported asmean±standard deviation.

Results

Enrichment for Sca-1⁺ Cells.

Table 7 shows the FASC analysis a representative of WBM cell populationand its corresponding enriched Sca-1⁺ cell population. Although WBMcells were isolated from the GFP transgenic mice, only ˜80% of theSca-1⁺ cells in this cell population expressed GFP (that is, were GFP⁺).Therefore, the relative percentage of the Sca-1⁺ cells that were GFP⁺versus those that were GFP⁻ during the enrichment process wasdetermined.

TABLE 7 Cell yield, recovery, and enrichment of Sca-1⁺ and/or GFP⁺ cellsduring the Sca-1⁺ cell enrichment process. Results are shown as mean ±S.D. for four replicate experiments. Number of cells per donor % Sca- %Sca- Fold mouse Recovery 1⁺-GFP⁺ 1⁺-GFP⁻ Total % Sca- enrichment of(×10⁶) (%) cells cells 1⁺ cells Sca-1⁺ cells Whole bone 116.0 ± 15.5(100)  4.7 ± 0.5. 1.2 ± 0.4  5.8 ± 0.7 (1) marrow Erythrocyte- 43.9 ±8.5   37.8 8.0 ± 1.7 2.8 ± 0.8 10.7 ± 0.9 2 lyzed cell subpopulationSca-1⁺-  3.0 ± 0.7    2.6 57.7 ± 9.9  16.6 ± 5.1  74.3 ± 5.3 13 enriched cell subpopulation

Table 7 summarizes the cell yield per donor mouse, recovery, percentSca-1⁺ (GFP⁺, GFP⁻, and total fractions), and fold of enrichment at eachisolation step of 4 independent experiments. The overall enrichment ofSca-1⁺ cells by this procedure was ˜13-fold. After the enrichment,approximately 80% of the Sca-1⁺ cells remained GFP⁺, demonstrating thatthis enrichment method does not discriminate GFP⁺-Sca-1⁺ cells fromGFP⁻-Sca-1⁺ cells.

Retroorbital Injection Vs. Tail Vein Injection.

Tail vein injection and retroorbital injection are two frequently usedroutes for delivering cells or test compounds into the circulation ofrecipient in rodents. Accordingly, the engraftment efficiency and thechimera levels between the retroorbital injection method and tail veininjection were compared at 8, 24, 32 and 36 weeks post-transplant andassessed for engraftment (FIG. 3A). At each test time point, every mice(6 of 6: 100%) in the retro-orbitally-injected group demonstratedsignificant engraftment, whereas only 11 out of 30 (37%) of the tailvein-injected mice showed evidence of engraftment. When the engraftmentin those 11 tail vein-injected mice was compared with that in theretro-orbitally-injected mice (excluding the 19 mice non-engrafted tailvein-injected mice), the retroorbital group showed a higher (72-82%)chimeric level than the tail vein group (56-66%) at each time point(FIG. 3A). The variation of the engraftment in the tail vein-injectedgroup was also significantly larger than that in the retroorbital group.Accordingly, Table 8 shows a 3- to 4-fold larger CV in the tail veingroup compared to the retroorbital group. The retroorbital injectionmethod is relatively safe to the host, as none of the mice injected byretroorbital injection suffered adverse effects to their eyes or werelost due to anesthesia.

TABLE 8 Comparison of coefficient of variation (CV) of the engraftmentbetween the tail vein-injected group and the retroorbitally-injectedgroup.* CV 8 24 33 37 40 weeks weeks weeks weeks weeks Tailvein-injected group 0.44 0.59 0.56 0.54 0.53 Retroorbitally-injected0.11 0.20 0.14 0.12 0.14 group *CVs were calculated from data presentedin FIG. 1. It was calculated as standard deviation divided by mean foreach time point.

Effect of Preconditioning on Engraftment Levels.

The combined effects of preconditioning and the use of W⁴¹/W⁴¹ recipientmice on engraftment at 4, 12 and 16 weeks were evaluated by comparingengraftment efficiencies in 4 recipient models (N=6 per group): 1)non-irradiated C57BL/6J wild type mice, 2) non-irradiated W⁴¹/W⁴¹ mice,3) sublethally (500 cGy) irradiated C57BL/6J wild type mice, and 4)sublethally irradiated W⁴¹/W⁴¹ mice. The results are shown in FIG. 3B.At all 3 time points, analysis by one-way ANOVA revealed statisticallysignificant differences between the four recipient models (p<0.01 in allcomparisons except at 12 weeks comparisons of wild type vs. W⁴¹/W⁴¹p<0.05). The engraftment in non-irradiated wild type recipients wasnegligible (0.54±0.18, 0.24±0.24 and 0.06±0.06% for 4, 12 and 16 weeks,respectively). Non-irradiated W⁴¹/W⁴¹ hosts had significantly increasedengraftment (7.0±5.7, 25.8±22.6 and 22.7±13.7%, respectively). Radiationpreconditioning of wild type mice markedly improved engraftment at eachtime point, (49.2±6.0, 57.2±7.8, and 54.0±7.2%, respectively). Thehighest level of chimerism was observed in the W⁴¹/W⁴¹ recipientsreceiving the sublethal irradiation (73.1±5.1, 82.6±7.3, 78.9±6.0%,respectively), indicating that the combination of preconditioning andthe use of genetically myelosuppressed recipient mice produced at leastan additive enhancement in engraftment. The enhanced engraftmentproduced by the combination persisted for up to more than one yearwithout significant reduction in chimera levels.

Effect of Delay of Injection on Long-Term Reconstitution.

Whether a 24-hour delay of the initiation of HSC transplantation afterthe sublethal irradiation affected long-term engraftment was alsotested. As seen in FIG. 3C, there was no significant difference inengraftment level between mice transplanted 4 hours vs. 24 hours postirradiation at any time point. Furthermore, engraftment was relativelyhigh and persisted up to 52 weeks (experimental endpoint). The 24 hoursdelay also did not lead to a significant increase in radiation-relatedmorbidity or mortality. These findings indicate that a 24-hour delaybetween sublethal irradiation and transplantation through retroorbitalinjection did not affect the short- or long-term engraftment efficiencyor cause significant increase in radiation-related morbidity and/ormortality.

Engraftment Efficiency of Genetically Altered Sca-1⁺ Cells.

Because the objective was to utilize this HSC transplantation strategyto develop a system for evaluating therapies for treating bone disordersand enhancing bone growth, it was important to ensure that ex vivomodification of Sca-1⁺ cells with viral transduction did not adverselyaffect engraftment efficiency or levels. To address this issue, Sca-1⁺cells of C57BL/6J wild type mice were transduced with a lentiviralvector expressing GFP. Forty-eight hours post-transduction, cells werestained with propridium iodide for assessment of cell viability and GFPexpression. The viral transduction caused significant cell death (81%),because a high MOI of viral vector was used. The GFP expression level ofthe transduced Sca-1⁺ cells was analyzed with FACS two and five daysafter transduction, which showed that approximately half of thetransduced cells (51% at 2 days and 49% at 5 days post-transduction)expressed the GFP trangene, indicating a ˜50% transduction efficiency(FIG. 4A).

An aliquot of 500,000 cells of the transduced cell preparation wereinjected into 24 sublethally irradiated W⁴¹/W⁴¹ recipient mice (N=12) 48hours post-transduction. At 9 weeks post-transplant, peripheral bloodwas collected from recipient mice and assayed for GFP mRNA expression byreal-time PCR and for percent GFP positive cells by FACS analysis, whichshowed greater than 80% GFP positive cells (FIG. 4B). Real-time PCRshowed detectable GFP expression in each recipient mouse but varied(mean GFP:GAPDH ratio was 91.9±54.3%). Engraftment was confirmed by FACSanalysis for GFP mononuclear blood samples. FIG. 4C shows a 50-60%long-term engraftment level in each of the recipient mice that lastedfor greater than 22 weeks.

The results discussed above demonstrate the efficacy of methods for theproduction of an improved murine Sca-1⁺ cell-based BMT system, involvingthe injection of a Sca-1⁺ cell enriched HSC subpopuation intosublethally irradiated, genetically myelosuppressed W⁴¹/W⁴¹ recipientmice through retroorbital vein 4 to 24 hours post-irradiation. Theseresults demonstrate that this strategy provided consistent (in everyrecipient mouse), long-term (up to greater than 52 weeks) engraftment ofvery high chimera levels (as high as 80%) with only a few hundredthousands of Sca-1⁺ donor cells, but yet caused minimal host morbidityand mortality. The engraftment levels in this study were estimated bymeasuring the relative amounts of GFP-expressing Sca-1⁺ donor cellsisolated from TgN-GFP transgenic mice in the recipients using FACS.Since approximately 80% of the Sca-1⁺ cells of the TgN-GFP transgenicmice are GFP⁺, the actual engraftment in this study is likely to besignificantly greater than 80% and probably even approaches full (100%chimera) engraftment. This study also showed that injections of Sca-1⁺cells derived from wild type C57BL/6J mice transduced with a lentiviralvector expressing the GFP marker gene yielded highly consistent (inevery test mouse) and high levels (50-60%) long-term engraftment. Theseresults demonstrate that the improved HSC-based BMT strategy can be usedas a system to evaluate osteogenic therapies, including, for example,gene and cell-based therapies.

Several features of this strategy contribute to achieving consistent,high levels of engraftment with minimal host morbidity and mortality.The strategy uses Sca-1⁺ cells rather than WBM cells as donor cells.WBM-based transplantation protocols require large numbers (e.g., severalmillions) of WBM to achieve appreciable engraftment (Uchida et al.,Blood 83:3758-3779, 1994). Intravenous injections of a large number ofcells are technically difficult and notoriously inconsistent. This largecell dose requirement also makes WBM transplantation less desirable forapplications such as gene therapy because it necessitates high viraltiters and/or high multiplicities of infection for adequate levels ofgene transfer. In contrast, Sca-1, a phosphatidylinositol-linked cellsurface glycoprotein, is a cell surface marker of primitive HSCs(Spangrude et al., Science 241:58-62, 1988; Uchida et al., Exp. Hematol.24:649-659, 1996), which have been shown to home to and engraft in boneafter intravenous injection (Krause et al., Cell 105:369-377, 2001;Uchida et al., Exp. Hematol. 24:649-659, 1996). Thus, one advantage ofthe Sca-1⁺ enriched subpopulation strategy over the WBM strategy is therequirement of a much lower number of donor cells for successfulengraftment. Consistent with this assumption, as few as about 300,000 to400,000 Sca-1⁺ cells yielded high level of long term engraftment,whereas it has been shown that several millions of WBM cells are neededfor successful engraftment (Uchida et al., Blood 83:3758-3779, 1994). Anadditional advantage of Sca-1⁺ cells over WBM cells is that this cellsubpopulation has been shown to provide both short-term radioprotection(Zhao et al., Blood 96:3016-3022, 2000; Okada et al., Blood80:3044-3050, 1992) and long-term, multi-lineage reconstitution (Osawaet al., Science 273:242-245, 1996; Zhao et al., Blood 96:3016-3022,2000; Okada et al., Blood 80:3044-3050, 1992) in lethally irradiatedmice. Although extra technical steps are needed for the isolation ofSca-1⁺ enriched subpopulation, recent advances in thefluorescent-assisted cell sorting technology and/or immunomagneticassisted cell isolation methods makes the isolation of Sca-1⁺ enrichedcell subpopulations relatively routine and cost-effective. As anexample, a 13-fold enrichment of Sca-1⁺ cells was obtained with a singleimmuno-magnetic column step.

The combined use of myelosuppressed W⁴¹/W⁴¹ mutant mice as therecipients and sublethal irradiation maximizes engraftment and, at thesame time, minimizes side effects due to irradiation preconditioning(Trevisan et al., Blood 88:4149-4158, 1996). Because the mechanismleading to myelosuppression by irradiation and that in W⁴¹/W⁴¹ mice isdifferent, low doses of irradiation along with the use of W⁴¹/W⁴¹recipient mice can be used to reduce host mortality and morbidity due tohigh dosage of radiation without significant reduction in engraftment.The results described above demonstrate that the use of W⁴¹/W⁴¹recipient mice and a sublethal radiation dose (500 cGy) in combinationproduced an additive enhancing effect on long-term engraftment withreduced host mortality and morbidity. These results are consistent withprevious studies that showed that irradiation preconditioningfacilitated the engraftment in W⁴¹/W⁴¹ recipient mice (Trevisan et al.,Blood 88:4149-4158, 1996).

Most prior BMT procedures require transplantation immediately afterirradiation preconditioning to reduce radiation-related mortality. Thisrequirement creates a significant time constraint on the experimentaldesign. The results described above demonstrate that a 24-hour delaybetween the time of transplantation and the time of the irradiation isfeasible in the current transplantation system because no significantdifferences in short- or long-term engraftment were observed in micewith a 4-hour or a 24-hour delay between irradiation and transplantationor in radiation-related morbidity and mortality in the host animals.This delay allows flexibility of the procedure with respect to thetiming of HSC transplantation, which adds a valuable advantage to thisSca-1⁺ cell-based BMT strategy in comparison to other strategies.

A single irradiation dose of 500 cGy was utilized because previousstudies indicated that this dose was the minimal radiation dose thatprovides sufficient myelosuppression to achieve high levels ofengraftment in the C57BL/6J allogenic model (Down et al., Blood77:661-669, 1991). However, lower or higher dosages of irradiation canbe used to further reduce host mortality and morbidity or to increaseengraftment without a significant increase in host mortality andmorbidity depending on individual experimental results.

The most commonly used and convenient route of cell delivery for murineBMT models is intravenous injection into the lateral tail vein. Due tothe small size of the lateral tail veins and their potential for venouscollapse, injection via tail vein can be technically difficult andhighly inconsistent, especially in the hands of a novice investigator.Injection into the retroorbital plexus is a convenient, relatively lessintrusive route for delivering test substances in murine models.Compared to tail vein injection, retroorbital injection is lesstechnically demanding and the results are less variable. Previous directcomparison between tail vein injection and retroorbital injection showedno differences in organ distribution or blood concentration of injectedmaterial (Price et al., Proc. Soc. Exp. Biol. Med. 177:347-353, 1984),indicating that retroorbital injection is a reliable delivery method forSca-1⁺ cells. Retroorbital injection resulted in greatly enhancedengraftment success (from 37% to 100%), higher engraftment levels, and asignificant reduction in variation as compared to the tail vein method.The retro-orbital injection method also led to engraftment in everyinjected mouse and a 3- to 4- fold reduction in intra-assay variationsas compared to the tail vein method. Since the power of an experiment isdirectly proportional to sample size and magnitude of the difference tobe detected and inversely proportional to the inherent variability ofthe observations (Samuals, Statistics for the Life Sciences, DellenPublishing Co., San Francisco, Calif., 1989), the reduction inintra-assay variability should result in an increase in experimentalpower and the ability to detect small differences, and/or decreasedsample size required. Additionally, no increased risk of fatality due toanesthesia was observed using this method (less than 1% of mice due toanesthesia or other complication). This risk can be further minimized byoptimizing the anesthesia dose according to the weight and strain of therecipient mouse.

The use of GFP transgenic mouse Sca-1⁺ cells as the donor cells affordsa convenient and reliable means to distinguish cells of donor from hostorigin and as such facilitates assessments of engraftment. This approachis feasible because both the TgN-GFP transgenic mice and the W⁴¹/W⁴¹recipient mice are of C57BL/6J background. The C57BL/6J mouse strainoffers several additional attractive features for use in studies ofHSC-based gene and cell therapy. First, this strain of mouse is widelyused in animal research, and a large body of genetic and biologicalinformation about this mouse strain is known. Second, numeroustransgenic, knockout, or mutant variations have been developed on thisstrain background and are available for use with the described strategyin assessing the functional role of the test compound, e.g., a testgene. Third, since it has been demonstrated that up to 99% of cells withmarrow reconstituting ability are contained within the Sca-1⁺ subset inC57BL/6J mice (Spangrude et al., Blood 82:3327-3332, 1993), the harvestof Sca-1⁺ cells from C57BL/6J background strain of mice provides cellpreparations with relatively high repopulating potential, which is animportant trait for BMT strategy.

Example 3 Sca-1⁺ Hematopoietic Progenitor Cell-Based Systemic GeneTherapy

Materials and Methods

Animals.

The TgN β-actin-eGFP (TgN-GFP) donor mice and wild-type C57BL/6J mousestrain were purchased from Jackson Laboratories (Bar Harbor, Me.).[TgN-GFP mice are GFP transgenic mice].

Bone Marrow Sca-1⁺ Cell Population Isolation.

Whole bone marrow (WBM) cells were harvested from TgN-GFP mice byflushing tibiae and femurs with sterile phosphate-buffered saline (PBS)supplemented with 0.5% bovine serum albumin (BSA) using a 26-g needleand syringe. Erythrocytes were removed by a 5-minute osmotic lysis atroom temperature using a solution of 155 mM NH₄C1, 10 mM KHCO₃ and 110μM Na₂EDTA, followed by rinsing with PBS. The cell preparation (1×10⁸cells per 0.8 ml of BSA-supplemented PBS) was then incubated withmagnetic microbeads conjugated with antibody specific for Sca-1 (0.2 mlbeads per 1×10⁸ cells) for 20 minutes at 4° C. and applied twice to anAutoMacs™ automated magnetic separation column according tomanufacturer's instruction (Miltenyi Biotec, Inc, Auburn, Calif.). Cellyields were measured by manual cell count of viable cells as determinedby trypan dye exclusion. Recovery was calculated by dividing the numberof cells counted after the lysis of erythrocytes and Sca-1⁺ enrichmentsteps by the number of WBM cells harvested. To assess enrichmentefficiency, aliquots of each cell preparation were incubated withPE-conjugated Sca-1 specific or PE-conjugated rat isotype controlantibody (Pharmingen, San Diego, Calif.) and analyzed for Sca-1 with aFACSCalibur System (BD Biosciences, San Jose, Calif.). The percentage ofSca-1⁺ cells was calculated by subtracting the value obtained with thePE-conjugated rat isotype control antibody from that obtained with thePE conjugated Sca-1 specific antibody.

Transduction of Sca-1⁺ Cells with MLV-Based Vectors.

The VSV-G pseudotyped MLV-based vectors expressing the modified FGF-2gene (pY-BMPFGFC2SC3N), the BMP2/4 hybrid gene (Gysin et al., Gene Ther.9:991-999, 2002; Peng et al., Mol. Ther. 4:95-104, 2001), or the eGFPgene (pY-GFP) were constructed as described previously (Peng et al.,Mol. Ther. 4:95-104, 2001). For the transduction, Sca-1⁺ cells wereplated in 6-well retronectin-coated plates at a density of 4×10⁶cells/well in IMDM media containing 10% fetal bovine serum (FBS), humanFlt-3 ligand, murine stem cell factor, murine IL-6, murine IL-1α andmurine IL-3 and 100 μM dNTPs. After 24 hours, the unconcentratedMLV-based viral stock was applied to the cells. After 8 hours the mediawas removed, and fresh media with cytokines and viral stocks werereapplied. After an additional 8 hours, the media was removed and freshmedia with cytokines (without virus) were again applied. Cells were usedfor transplantation within 12-24 hours after transduction.

Transplantation.

Two weeks before and 2 weeks after the irradiation procedure, recipientmice were provided sterile food and autoclaved, acidified water (pH2.0-2.5) containing 50 mg/L neomycin sulfate and 13 mg/L polymixin Bsulfate. Recipient mice were preconditioned by total body irradiationwith using a ⁶⁰Co source delivering a single radiation dose of 500 cGyat a rate of 80 cGy per minute. The transduced donor Sca-1⁺ cells werethen transplanted into anesthetized (with 62 mg/kg ketamine and 12 mg/kgxylazine) recipient mice 4 hours post-irradiation via retro-orbitalinjection. An aliquot of 500,000 Sca-1⁺ cells (in 30 μl sterile saline)were injected into each W⁴¹/W⁴¹ recipient mouse via retroorbital plexuswith a 30-g, ½-inch needle. At the endpoint, mice were anesthetized,decapitated, and whole blood (˜700 μl) was collected from each mouse.The percent eGFP-expressing cells (to monitor engraftment) weredetermined by FACS analysis. Serum FGF-2 protein levels were determinedby ELISA using a commercial kit (R&D Systems, Minneapolis, Minn.).Aliquots of serum samples were kept frozen at −80° C. for skeletalalkaline phosphatase (ALP), PTH, and calcium assays. Serum ALP andcalcium were performed by the Hitachi 912 Clinical Chemistry analyzer.The mean inter- and intra-assay CV for serum calcium assay was <2% withthe sensitivity of 0.2 mg/dL and measuring range of 0.2-18 mg/dL. SerumPTH was measured by ELISA (PTH Immunoassay Kit, ALP-Co™). The boneextract ALP activity was assayed by a colorimetric assay as describedpreviously (Farley et al., Calcif. Tissue Int. 50:67-73, 1992). BMP-4level in CMs and cell extracts were estimated from an immunoblot assaysby comparing its relative densitometric intensity with that of knownamounts of BMP-4 recombinant protein.

Analysis of Engraftment of Transduced Cells.

At various times post transplantation of pY-GFP-transduced Sca-1⁺ cells,peripheral blood was collected via the lateral tail vein. Erythrocyteswere lysed and FACS analysis was performed for percent eGFP-expressingdonor cells.

Immunohistochemical Staining of eGFP-Expressing Cells.

Donor cells from TgN-GFP transgenic mice were identified byimmunohistochemical staining of eGFP to avoid interference fromautofluorescence in bone sections. Briefly, thin (5 μm) frozenlongitudinal sections of right femurs of recipient mice were prepared bycryostat, immediately immersed in 100% methanol for 20 min at roomtemperature. The sections were then re-hydrated and rinsed with PBS.Non-specific antigens were inactivated by exposing the sections to 3%H₂O₂ for 10 min at room temperature. The sections were then rinsed withPBS 4-5 times and the non-specific antibody binding sites were blockedby incubating the sections with 20% normal rabbit serum for 30 min at37° C. The sections were then incubated with the chicken anti-GFPantibody (1:100 dilution) followed by incubation with the biotinylatedanti-chicken IgG secondary antibody (1:250 dilution) using the VentanaES immunostainer (Ventana, Tucson, Ariz.), accordingly themanufacturer's instruction. The immunostained sections werecounterstained with hematoxylin for 30 sec, air-dried, and covered withcoverslips. Osteoblasts were identified as a palisade of largebasophilic cells directly lining the bone surface. Osteoclasts wereidentified as large multinucleated, irregularly-shaped cells with astriated perimeter zone of attachment (ruffled border) to the bonesurface (resorption pits).

pQCT Measurements.

Femurs of each mouse were dissected, formalin-fixed, and stored in PBS(containing 0.5% sodium azide). Cross-sectional and volumetric boneparameters were measured using an XCT 960M with XCT software version5.40 in a multispecimen holder designed for the XCT 960M. Voxel size wasreduced to 0.07 mm for the analysis. The bone scans were analyzed withtwo different outer threshold settings to separate bone from softtissue. A threshold setting of 630 mg/cm³ was used to determine boneareas and surfaces. A second analysis with a threshold of 230 mg/cm³ wasused to determine mineral content. These thresholds were selected toyield area values consistent with histomorphometrically derived values.The measured parameters included total, trabecular, and corticalvolumetric bone mineral density (vBMD), cortical thickness, andperiosteal and endosteal circumferences. Total, cortical, and trabecularvBMD values were calculated by dividing the total, cortical, andtrabecular mineral content by the total, cortical, and trabecular bonevolume, respectively, and expressed as mg/cm³.

Bone Histomorphometry.

Bone histomorphometry was performed in the femurs. After pQCTmeasurements, the left femurs were decalcified in 14% EDTA for 2 weeksat 4° C., dehydrated in ethanol, infiltrated, and embedded in paraffinwax. Longitudinal serial sections (5 μm in thickness) were stained withhematoxylin and eosin (H&E stain) or Mallory's trichrome stain (fordetection of collagen). To measure bone resorption parameters, serialsections were stained for tartrate-resistant acid phosphatase (TRAP)activity as described previously (Boyce et al., Endocrinology136:5751-5759, 1995). The TRAP stained sections were counterstained withmethyl green and light green SF yellowish.

Right femurs were embedded into methylmethacrylate (MMA) for assessmentof bone mineralization parameters. Briefly, the right femurs weredehydrated with 95% and 100% ethanol each for two days. The bonespecimens were then infiltrated with a mixture containing 85 ml of MMA,10 ml of glycolmethacrylate, 5 ml of dibutylphthalate, 5 ml ofpolyethylene-glycol, and 0.9% benzoylperoxide for 4-6 days. Thissolution was changed every 2-3 days. Bone specimens were then placed inplastic molding cups (6×12×5 mm) and orientated at a position with theanterior part of the femur facing down. Polymerization was initiatedwith 1% JB4 solution B (Polysciences, Inc., Warrington, Pa.). After 5-10min of vacuum, bone specimens were carefully transferred into a gas jarand flushed with nitrogen for 5-10 min. The gas jar was then sealed andplaced in cold for 2 days for polymerization. Serial sections (5-10 μmin thickness) were stained with Goldner's trichrome stain.

Statistical Analysis.

Comparisons of differences were performed using the two-tailed,two-sample with equal variances, independent t-test, one-way ANOVA, andlinear regression. Results were considered significant when p<0.05. Alldata are reported as mean±standard error of the mean.

Preparation of the MLV.tet.e.GFP and MLV.tet.FGF* Vectors.

The doxycycline-inducible MLV-based vector system (rtTA-GYT) was basedon the tet-on inducible system reported by Das et al., J Biol Chem 279,18776-18782, 2004). The system utilized herein has 3 additionalmutations form rtTA (mutations at S 12G, F86Y and A209T from rtTA-S2)and exhibits better inducibility than the original clones used by Das etal.). The rtTA-GYT and the tetracycline controlled promoter (TetO-P)were incorporated into the MLV.eGFP and MLV.FGF2* vectors to generateMLV.tet.eGFP and MLV/tet.FGF2* respectively.

Results

Transduction of Murine Sca-1⁺ Cells with MLV-Based Vectors.

To demonstrate that Sca-1⁺ cells can be transduced with MLV-basedvectors, Sca-1⁺ cells isolated from wild-type C57BL/6J mice weretransduced with a pY-GFP MLV-based vector and the percent ofGFP-expressing Sca-1⁺ cells was measured 48-hr after transduction toassess transduction efficiency. The transduction efficiency wasconsistently greater than 50%. The amounts of FGF-2 protein was alsomeasured in the conditioned media (CMs) of Sca-1⁺ cells transduced withthe MLV vector expressing the modified FGF-2 gene (pY-BMPFGFC2SC3N) 72hrs after transduction and compared to those in the CMs of Sca-1⁺ cellstransduced with the MLV vector expressing the unmodified FGF-2 gene(pY-FGF) or the marker gene, β-galactosidase (pY-β-gal). The averageamount of FGF-2 secreted by the pY-β-gal-transduced Sca-1⁺ cells wasbarely detectable (˜2 ng/10⁶ cells/24 hrs) and that of pY-FGF-transducedSca-1⁺ cells was low (˜18 ng/10⁶ cells/24 hrs). In contrast, the amountof FGF-2 secreted by pY-BMPFGFC2SC3N-transduced Sca-1⁺ cells was >320ng/10⁶ cells/24 hrs (>15-fold more than that by pY-FGF-transducedcells). Sca-1⁺ cells transduced with pY-BMP2/4 vector secreted ˜10ng/10⁶ cells/24 hrs mature BMP-4 protein (estimated by the immunoblotassay).

Evidence that Transplanted Sca-1⁺ Cells can Contribute Donor Progenitorsto the Osteoblastic Lineage.

To assess whether the engrafted Sca-1⁺ enriched cell population can bedifferentiated into bone cells at the endosteal bone surface,eGFP-expressing Sca-1⁺ cells isolated from the TgN-GFP transgenic donormice were transplanted into sub-lethally W⁴¹/W⁴¹ recipient mice, and thebone marrow cavity of recipient mice for GFP-expressing bone cells (byimmunohistochemical staining) was analyzed one yearpost-transplantation. Frozen sections of mouse femurs receivingGFP-expressing Sca-1+ donor cells were immunostained for GFP one yearpost-transplantation. There were numerous osteoblast- andosteoclast-like cells that stained strongly for GFP on the endostealbone surface. While it is expected that Sca-1⁺ cells can differentiateinto osteoclastic cells, since osteoclastic cells are derived from cellsof the hematopoietic lineage, it is surprising to note that the donorGFP-expressing Sca-1⁺ cells most predominantly localized to the marrowcavity along the endosteal surface can also be differentiated intoosteoblastic cells. These findings indicate that the donor Sca-1⁺enriched cell population can contribute osteoblastic progenitors to therecipient mice.

Retro-Orbital Delivery of MLV-Transduced Sca-1⁺ Cells into Sub-LethallyIrradiated W⁴¹/W⁴¹ Recipient Mice LED to Engraftment.

To confirm the feasibility of using the Sca-1⁺ enriched cell populationto deliver the modified FGF-2 gene to bone marrow cavity, approximately500,000 Sca-1⁺ cells transduced with the modified FGF-2 MLV vector(pY-BMPFGFC2SC3N) were transplanted via retro-orbital plexus into 8sub-lethally irradiated female W⁴¹/W⁴¹ recipient mice. For assessment ofengraftment and as a control for comparison, the same number of Sca-1⁺cells transduced with the pY-GFP marker gene was also transplanted into8 sub-lethally irradiated female W⁴¹/W⁴¹ recipient mice. At 10 and 12weeks post-transplantation, peripheral blood was collected and percentGFP-expressing cells were determined by FACS analysis. All mice weresacrificed at 12 weeks and the percent of GFP-expressing bone marrowcells was determined (Table 9). The average transduction and engraftmentin this experiment (as reflected by percent GFP-expressing cells in theperipheral blood) was ˜35%. The percent of GFP-expressing bone marrowcells in the engrafted recipients was about one-third to one-half lowerthan that in peripheral blood. As anticipated, the mice that receivedthe modified FGF-2 vector-transduced Sca-1⁺ cells did not havesignificant numbers of GFP-expressing cells (background levels).

TABLE 9 Mean percent eGFP-expressing cells in the peripheral blood andin bone marrow of recipient mice at 10 and 12 weekspost-transplantation.* (Mean ± SEM, n = 8). Bone Marrow Peripheral BloodCells Cells Treatment Group Week 10 Week 12 Week 12 GFP-expressingSca-1⁺ 35.5 ± 4.3%  34.1 ± 4.1% 11.6 ± 2.4% cells transplanted GroupModified FGF-2- 0.40 ± 0.24% 0.32 ± 0.15  0.44 ± 0.11  expressing Sca-1⁺cells transplanted Group *% eGFP-expressing cells were determined byFACS analysis as described in Methods after erythrocytes were removed byosmotic lysis.

To confirm that Sca-1⁺ cells transduced with the modified FGF-2 vectoralso engrafted, the serum FGF-2 levels in the recipient mice weremeasured 10 or 12 weeks post-transplantation (Table 10). The averageserum FGF-2 level in the pY-GFP-transplanted control mice was 35.6pg/ml, which was not significantly different from that in untreatedcontrol mice. In contrast, the average serum FGF-2 level in the modifiedFGF-2 vector-transplanted recipient mice was ˜100-fold higher than thatin pY-GFP-transplanted control mice. The serum level of individualrecipient mouse was variable (ranging from 77 pg/ml to 6,400 pg/ml) witha within-group coefficient of variance (CV) of 0.98. In spite of thewithin-group variation, the increase in serum FGF-2 level was highlysignificant (p=0.014). These findings are consistent with the previousfindings that retroviral transduction does not limit engraftment (Kanget al., Hum. Gene. Ther. 12:1663-1672, 2001).

TABLE 10 Serum FGF-2, ALP, and osteoclacin levels and tibial ALP levelsin the recipient mice 10- or 12-weeks post-transplantation.^(a) (Mean ±SEM, n = 8). Serum Serum Tibial ALP Serum FGF-2^(b) ALP Osteocalcin(mU/mg dried Treatment Group (pg/ml) (mU/ml) (ng/ml) bone)GFP-expressing Sca-1⁺ 35.6 ± 2.1 177.8 ± 8.6  8.05 ± 0.18  0.149 ± 0.049cells transplanted (28-43)^(c)   (145-212)^(c) (4.2-12.8)^(c)(0.006-0.307)^(c)  group Modified FGF-2- 2,502.9 ± 929.4^(d) 271.9 ±37.3^(e) 9.91 ± 2.67^(f)  5.189 ± 1.724^(g) expressing Sca-1⁺ cells(77-6,400)^(c) (166-389)^(c) (3.5-24.8)^(c) (0.451-13.128)^(c)transplanted group ^(a)Three of the eight pY-BMPFGFC2SC3N-transplantedrecipient mice were noticeably ill and were euthanized at week 10. Therest of the mice were euthanized at week 12. ^(b)Most of thepY-BMPFGFC2SC3N-transplanted recipient mice had serum FGF-2 levelshigher than the upper limit of the ELISA assay (>640 pg/ml). Thereported values for these mice were extrapolated from the standardcurve. ^(c)Ranges. ^(d)p = 0.014, two-tailed Student's t-test. ^(e)p =0.021, two-tailed Student's t-test. ^(f)p = n.s., two-tailed Student'st-test. ^(g)p = 0.022, two-tailed Student's t-test.

To evaluate the time-course of engraftment of modified FGF-2vector-transduced Sca-1⁺ cells, the pY-BMPFGFC2SC3N-transduced Sca-1⁺cells (500,000 cells) and the pY-GFP-transplanted Sca-1⁺ cells were eachtransplanted into 8 sub-lethal female W⁴¹/W⁴¹ recipient mice. The serumFGF-2 level of each mice at 8-, 10-, 12- and 14-weekpost-transplantation was measured and is shown in FIG. 5A. Earlier timepoints were not performed because previous studies have indicated thatSca-1⁺ cells engraft over a period of 4 to 8 weeks (Tomita et al., Blood83:939-948, 1994). With the exception of the 14-week time point, theserum FGF-2 level of the FGF-2 group at each time increased with timeand was significantly higher than that of the GFP control group.

In a separate experiment, the engraftment of pY-BMP2/4-transduced Sca-1⁺cells was assessed by measuring the presence of the BMP2/4 gene ingenomic DNA of peripheral blood of recipient mice using a PCR-basedassay. The relative level of BMP2/4 gene incorporated in DNA ofrecipient mice was very similar to that of β-gal gene in recipient micetransplanted with pY-β-gal-transduced Sca-1⁺ cells. This suggests thatthe pY-BMP2/4-transduction did not significantly affect the engraftmentof pY-BMP2/4-transduced Sca-1⁺ cells.

Transplantation of Modified FGF-2-Expressing Sca-1⁺ Cells InducedEndosteal Bone Formation in Recipient Mice.

Table 10 shows that the recipient mice of the modified FGF-2vector-transduced Sca-1⁺ cells at 10- to 12-week post-transplantationshowed a significant (p=0.021) increase in serum ALP activity [abiochemical marker of bone formation (Farley et al., Metabolism35:563-571, 1986)] compared to recipient mice receiving thepY-GFP-transduced cells. Serum osteocalcin level (another bone formationmarker) in the FGF-2 group at 10- to 12-week was also higher than thatin the GFP control group (9.91±2.67 vs. 8.05±0.18 ng/ml). To confirm anincrease in bone ALP levels in the FGF-2 group, the ALP activity intibial extracts was measured, normalized against dried bone weight(Table 10). The FGF-2 group showed a 34-fold elevation (P=0.022) in thetibial ALP activity compared to the GFP control group. A similarincrease was seen with the tibial ALP specific activity (normalizedagainst the bone extract protein). The elevated levels of serum and bonebiochemical markers of bone formation in the FGF-2 group suggest anincrease in bone formation.

The serum and tibial bone extract ALP activities in eight recipient micetransplanted with pY-BMP2/4-transduced Sca-1⁺ cells were determined at14-weeks post-transplantation and compared to those in eight recipientmice transplanted with pY-GFP-transduced Sca-1⁺ cells. Neither the serumALP level (183.6±10.8 mU/ml in the pY-GFP group vs. 174.5±11.7 mU/ml inthe pY-BMP2/4 group, p=N.S.) nor the tibial extract ALP level(0.079±0.005 mU/mg dried bone in pY-GFP group vs. 0.079±0.008 mU/mgdried bone in pY-BMP2/4 group, p=N.S.) was significant different betweenthe pY-BMP2/4 group and the pY-GFP control group. This indicates that,in contrast to the modified FGF-2 treatment, the BMP-2/4 treatmentfailed to produce an osteogenic effect in this system.

To evaluate the relationship between the increase in serum FGF-2 levelsand bone ALP activity, the tibial ALP activity was plotted against theserum FGF-2 level of the 8 mice in the FGF-2 group (FIG. 5B). There wasa highly significant correlation (r=0.70, p<0.004) between the twoparameters, showing that the increase in the bone ALP activity (boneformation) is associated with the increase in the serum FGF-2 level ofthe recipient mice.

Various parameters of bone mineral density (BMD) and bone mass weremeasured in recipient mice using pQCT. The combined results of severalexperiments are shown in Table 11. At 10- to 14-weekpost-transplantation (end-point), there was a significant increase intotal vBMD (14%) and trabecular vBMD (2.5-fold), but not cortical vBMD,in the FGF-2 group compared to the GFP control group. The small (4%)decrease in endosteal circumference in the FGF-2 group compared to theGFP control group is consistent with an increase in endosteal boneformation. These findings are consistent with a previous studydemonstrating that FGF-2 protein administration in the rat induced boneformation primarily at trabecular bone sites (Mayahara et al., GrowthFactors 9:73-80, 1993). The FGF-2 group also showed a small (13%) butstatistically significant decrease in cortical thickness and asignificantly shorter (8%) periosteal circumference. These findingssuggest that the transplantation of modified FGF-2-expressing Sca-1⁺cells, while led to increase in endosteal trabecular bone formation, mayhave caused bone loss at cortical and/or periosteal bone sites.Consistent with a lack of osteogenic response by the systemic BMP2/4 exvivo gene transfer approach, Table 11 also shows that the BMP2/4treatment produced no significant effect on any of the test pQCT boneparameters compared to the pY-GFP control group.

TABLE 11 Effects of transplantation of Sca-1⁺ cells transduced with theMLV-GFP vector, the MLV-modified FGF-2 vector or the MLV-BMP2/4 vectoron the cross-sectional and volumetric bone parameters of the femurs ofrecipient mice at week 14 post-transplantation. (Mean ± SEM, n = 16).Bone Parameters GFP group FGF-2 group BMP-4 group Total vBMD (mg/cm³)742.0 ± 11.7  847.0 ± 68.9* 742.1 ± 9.9  Trabecular vBMD (mg/cm³) 229.0± 11.7    573.0 ± 113.1*** 270.1 ± 15.5  Cortical vBMD (mg/cm³) 1,099.0± 18.4   1,115.0 ± 49.9   1,092.7 ± 23.9   Cortical thickness (mm) 0.292± 0.007  0.254 ± 0.021* 0.293 ± 0.007 Periosteal circumference (mm) 5.20± 0.09  4.80 ± 0.17** 5.22 ± 0.07 Endosteal circumference (mm) 3.36 ±0.08 3.22 ± 0.09 3.37 ± 0.05 *p < 0.05, **p < 0.01, and ***p < 0.001.

Linear regression analysis between the serum FGF-2 level and each of themeasured pQCT parameters revealed a highly significant positivecorrelation between serum FGF-2 and total vBMD (r=0.37, p<0.05) andbetween serum FGF-2 and trabecular vBMD (r=0.54, p<0.002). Conversely,serum FGF-2 inversely and significantly correlated with corticalthickness (r =−0.51, p<0.005), periosteal circumference (r=−0.62,p<0.0004), and endosteal circumference (r=−0.47, p<0.01).

Effects of Transplantation of Modified FGF-2-Expressing Sca-1⁺ Cells onGrowth and the Size of Major Organs.

To determine whether the transplantation of modified FGF-2-expressingSca-1⁺ cells had adverse effects on the growth of recipient mice, thebody weight of each mouse was measured at week 7 and week 12. There wasno significant difference in the growth rate between the FGF-2 group andthe GFP control group, indicating that the elevated circulating FGF-2did not have an adverse effect on the growth of recipient mice. To testwhether the transplantation of modified FGF-2-transduced Sca-1⁺ cellshas an adverse effect on the general health of the recipient mice, theweight of major internal organs was measured at the time of thesacrifice (week 14). Table 12 shows that the increased FGF-2 expressionin the FGF-2 group did not significantly affect the weight of the heart,kidneys, lung, and intestine/stomach of recipient mice compared to theGFP control group at 14 weeks post-transplantation. Conversely, theweight of the spleen and the liver was each significantly increased,indicating an enlargement of these organs. It is especially true for thespleen, since the average weight of spleen in the FGF-2 group wasincreased 4.5-fold. However, with the exception of the enlarged spleen,a gross examination of these organs at the time of dissection did notreveal obvious abnormalities.

TABLE 12 Effects of transplantation of FGF-2 expressing Sca- 1⁺ cells onthe weight of various internal organs of recipient mice at 14-weekpost-transplantation. Internal organ (gm) GFP group FGF-2 group p-value*Heart 0.138 ± 0.008 0.133 ± 0.011 n.s. Kidneys (average) 0.165 ± 0.0050.169 ± 0.006 n.s. Spleen 0.082 ± 0.007 0.370 ± 0.057 <0.0001 Liver1.203 ± 0.112 1.495 ± 0.053 <0.05  Lung 0.367 ± 0.021 0.364 ± 0.040 n.s.Intestine/Stomach 2.996 ± 0.104 3.221 ± 0.212 n.s. *Two-tailed student'st-test.

Effects of Transplantation of Modified FGF-2 Expressing Sca-1⁺ Cells onthe Histomorphometry of Long Bones in Recipient Mice.

Gross examination of the long bones during dissection revealed anunusual gross appearance in recipient mice that had very high serumFGF-2 levels (>4,000 pg/ml). Femurs were dissected from a representativerecipient mouse receiving transplantation with GFP-transduced Sca-1+cells and from a representative recipient mouse receiving the modifiedFGF-2 MLV vector, which showed a very high (>4,000 pg/ml) serum FGF-2level. The bones were dissected from recipient mice 10-weekpost-transplantation. Instead of the characteristic maroon color of longbones (representing the red marrow), the femurs of mice with high serumFGF-2 levels were white (without the appearance of the red marrow).Without being bound by theory, the lack of bone marrow within the marrowcavity explains the lack of the characteristic maroon color of redmarrow in these few bones. Since the spleen and, to some extent, theliver are the extramedullary sites for blood cell production, the lackof bone marrow also helps to explain the enlargement of the spleen andthe liver in recipient mice receiving transplantation of modifiedFGF-2-expressing Sca-1⁺ cells.

To further evaluate the de novo endosteal bone formation,histomorphometric analysis was performed on the femurs. One femur ofeach mouse was demineralized and embedded in paraffin. Seriallongitudinal sections were stained with hematoxylin and eosin (H&E) forvisualization of tissue structure and also with Mallory's stain forcollagenous fibers. The contralateral femur was embedded inmethylmethacrylate. Longitudinal serial sections were then stained withGoldner's stain for matrix mineralization. The mice were arbitrarilydivided into three groups according to their serum FGF-2 level: 1) thosewith basal serum FGF-2 levels (<35 pg/ml) [the GFP control mice]; 2)those with intermediate serum FGF-2 levels (50-200 pg/ml); and 3) thosewith high serum FGF-2 levels (>1,000 ng/ml). The H&E stainedmid-diaphyseal sections confirm that the dark purplish-stained bonemarrow seen in the basal serum FGF-2 control group was completelyreplaced by the pinkish bone-like tissues in the high serum FGF-2 group.In the intermediate serum FGF-2 diaphyseal sections, there was alsoevidence that a significant amount of bone marrow was replaced withnewly formed bony tissues, some of which even extended to endostealsurfaces. While some of this newly formed bone appeared to be wovenbone, there was a considerable amount of normal, lamellar bone. TheMallory's stained sections shows that the pinkish stained areas withinthe marrow cavity of the groups of mice with intermediate or high serumFGF-2 seen in H&E sections also stained strongly for collagen fibers,confirming that the newly formed tissues within the bone marrow cavitywere bony tissues. When the un-demineralized sections were stained withGoldner's stain, the bones of the control mice and those of mice whichhad intermediate serum FGF-2 levels each showed only mineralized boneswithout detectable unmineralized reddish stained bony tissues. Theamount of mineralized bone was much more in the group with intermediateserum FGF-2 than the control group. In contrast, there was abundance ofunmineralized osteoid-like tissues in mice with high serum FGF-2,especially in the marrow cavity where newly formed bony tissues werelocated. These histomorphometric observations confirm thattransplantation of Sca-1⁺ cells expressing modified FGF-2 inducedmassive de novo endosteal bone formation, and suggest that the boneformation was so robust that the levels of minerals present are notoptimal to fully mineralize the newly formed bone, leading toosteomalacia.

Evidence that Excessive Endosteal Bone Formation in Mice with High SerumFGF-2 Developed, Hypocalcemia, Secondary Hyperparathyroidism, andIncreased Bone Resorption.

Consistent with the premise that dietary calcium is insufficient to meetthe demand of the mineralization of the massive endosteal boneformation, the serum total calcium level of mice transplanted withSca-1⁺ cells expressing the modified FGF-2 was significantly lower thanthat in mice transplanted with Sca-1⁺ cells expressing the GFP controlgene at the time of the sacrifice at 12 week post-transplantation(9.82±0.31 mg/dl vs. 10.66±0.21 mg/dl, mean±SEM, n=8, p=0.035). Althoughthe serum ionized calcium level was not measured in these mice, thissuggests that the massive endosteal bone formation in the FGF-2 groupled to hypocalcemia.

A well known consequence of hypocalcemia is secondaryhyperparathyroidism. Accordingly, the serum levels of the mice weremeasured at the time of sacrifice (12 weeks post-transplantation) andthe serum PTH levels were stratified into 4 groups according to theserum FGF-2 level: 1) ≦35 pg/ml (control, basal level), 2) 36-199 pg/ml,3) 200-1,000 pg/ml, and 4) >1,000 pg/ml. FIG. 19 shows that there was ahighly significant (p<0.001, one-way ANOVA), dose-dependent increase inserum PTH level with respect to serum FGF-2 level in the recipient mice.Except for the 36-199 pg/ml group, the increase in serum PTH in eachsubgroup was significantly (p<0.001) different from the control group.These findings confirm that the robust increase in endosteal boneformation in the FGF-2 mice led to secondary hyperparathyroidism.

A response to secondary hyperparathyroidism is an increased boneresorption. As an initial test for this possibility, the number ofTRAP-positive multinucleated osteoclasts was measured on demineralizedparaffin-embedded femoral sections. Immunohistochemical staining fortartrate-resistant acid phosphatase (TRAP) was evaluated in sections offemurs from 3 representative mice transplanted withpY-BMPFGFC2SC3N-transduced Sca-1+ cells that exhibited different levelsof serum FGF-2. Recipient mice with high serum levels of FGF-2 had moreTRAP-positive osteoclasts on bone surfaces than mice with intermediateserum FGF-2 levels, which had more TRAP-positive osteoclasts on bonesurfaces than those with normal serum FGF-2 levels.

Increased trabecular bone formation at endosteal bone sites is essentialfor an effective treatment of osteoporosis and related musculoskeletaldiseases. An increase in the amount of trabecular bone at the endostealsurfaces increases bone strength and reduces the risk of fractures. Theresults described above show that Sca-1⁺ hematopoietic stem andprogenitor cells have propensity to home to and engraft in the bonemarrow cavity (Spangrude et al., Blood 85:1006-1016, 1995), increasingthe efficacy of bone marrow transplantation for osteogenic therapies.

Thus, Sca-1⁺ hematopoietic stem cells and progenitor cells can be usedas the vehicle for osteogenic factors, for example, osteogenic growthfactors, such as FGF-2 to promote endosteal bone formation. Suitableosteogenic growth factors are those, like FGF-2 that not only stimulatesbone formation but also promotes hematopoietic stem cell renewal. FGF-2promotes stem cell renewal without significantly inducing theirdifferentiation (Kashiwakura et al., Brit. J. Haematol. 122:479-488,2003; Kashiwakura et al., Leuk. Lymphoma 46:329-333, 2005). It hasrecently been reported that FGF-2 enhances undifferentiatedproliferation of stem cells (He et al., Nat. Genet. 36(10):1117-1121,2004). Moreover, FGF-2 is a known potent angiogenic agent.Vascularization of endosteal bone is essential to support endosteal boneformation. The lack of angiogenic effect of such factors as BMP2/4 mayalso partly contribute to the lack of enhanced endosteal bone formationby BMP-2/4 in this system. Therefore, FGF-2 and bioactive analogs ofFGF-2 are suitable bone growth factor transgenes to promote endostealand/or trabecular bone formation.

Intravenous injection of a Sca-1⁺ enriched cell population transducedwith MLV-based vector expressing a modified FGF-2 gene in sublethallyirradiated myelosuppressed W⁴¹/W⁴¹ recipient mice led to long-termengraftment of FGF-2-expressing cells in the bone marrow cavity. Theengrafted cells retained their ability to express large amounts of FGF-2protein as the average serum FGF-2 level of recipient mice was >20-foldhigher than that of control mice. These findings not only confirm theprevious suggestion that the viral transduction process did notsignificantly impede the engraftment of Sca-1⁺ cell (Deola et al., Hum.Gene Ther. 15:305-311, 2004; Hall et al., to be submitted, 2005), butmore importantly, shows for the first time that intravenoustransplantation of Sca-1⁺ cells expressing the modified FGF-2 gene ledto a marked increase in bone ALP activity and a large increase in totaland trabecular vBMD in recipient mice, which indicate a largeenhancement in bone formation. The observations of the pQCT and bonehistomorphometric evaluations of femurs of recipient mice indicatingthat the enhanced bone formation occurred primarily at trabecular boneson the endosteum are consistent with the previous finding that FGF-2stimulated primarily trabecular bone formation on the endosteum withoutincreasing periosteal and/or cortical bone formation (Mayahara et al.,Growth Factors 9:73-80, 1993).

The results described above demonstrate certain additional benefits ofusing Sca-1⁺ hematopoietic progenitor cells as the target cellpopulation and FGF-2 to promote endosteal bone formation. For example,two recent studies have provided strong evidence for thetrans-differentiation of hematopoietic progenitor cells into cells ofosteoblast lineage (Olmsted-Davis et al., Proc. Natl. Acad. Sci. USA100:15877-15882, 2003; Dominici et al., Proc. Natl. Acad. Sci. USA101:11761-11766, 2004). In the present study, a different experimentalapproach from the previous studies was utilized, e.g., using Sca-1⁺cells isolated from the TgN-eGFP transgenic mice to follow the fate ofdonor cells in recipient mice. In this system, GFP-expressingosteoblasts (derived from the GFP-expressing donor Sca-1⁺ cells) wereobserved at the endosteal bone surface of recipient mice. Thisdemonstrates that some of the donor Sca-1⁺ cells havetrans-differentiated into osteoblasts. Thus, the use of Sca-1⁺hematopoietic stem and/or progenitor cells as target cells has anadvantage over other target cells in that the trans-differentiationenhances the commitment of transduced cells into cells of osteoblasticlineage, which is involved in enhanced endosteal/trabecular boneformation.

In addition, FGF-2 treatment promoted hematopoietic cell expansion andstem cell renewal, and also stimulated the formation of an adherentstromal cell layer in human bone marrow cultures (Kashiwakura et al.,Brit. J. Haematol. 122:479-488, 2003; Kashiwakura et al., Leak. Lymphoma46:329-333, 2005). Accordingly, the expression of FGF-2 transgene inthese cells further enhances the renewal of FGF-2 expressinghematopoietic stem cells and the proliferation and commitment ofengrafted FGF-2-expressing hematopoietic cells into cells of osteoblastlinkage, which further enhances bone formation.

Intravenous injection of the Sca-1⁺ enriched cell population transducedwith the MLV-modified FGF-2 vector in sublethally irradiated W⁴¹/W⁴¹mice not only promoted endosteal/trabecular bone formation, but in factled to a massive de novo endosteal/trabecular bone formation within themarrow cavity. Bone histomorphometric analysis of the femurs ofrecipient mice reveals that the enhanced endosteal bone formation inthose several recipient mice that had a very high serum FGF-2 level(e.g., >4,000 pg/ml) was so robust that the entire marrow cavity wascompletely filled up with newly formed bony tissues. Consequently, thesefindings indicate that this transplantation approach and the use of themodified FGF-2 gene provided a very powerful means to increase de novoendosteal bone formation and promote division and differentiation ofendosteal/trabecular bone cells. For example, with respect to treatingosteoporotic patients, who are mostly elderly patients, it is useful toincrease bone formation in a relatively short period of time to increasebone strength in order to avoid further fractures. Accordingly, thestrategy disclosed herein overcomes the major deficiency in the field ofosteoporosis treatment, that is, the ability to produce a sufficientamount of bone in a realistic timeframe. Consequently, strategydescribed herein is an ideal form of therapy for the treatment ofostoporosis and other conditions that require rapid bone growth.

The distribution and the location of the increased endosteal/trabecularbone formation is an important issue. Ideally, in order to realizemaximal effects on the bone strength, the bone formation should beincreased at sites where the increased bone formation is needed themost, that is, at bone sites where the strains and stresses imposed bynormal physical activities are the greatest and at the site offractures. Mechanical loading is perhaps the most importantphysiological feedback regulator of bone remodeling and the skeletalarchitectural integrity, as there is a strong relationship between theorientation of trabecular architecture and the assumed principal stressdirections resulting from normal loading boundary conditions.Accordingly, it has been well known that mechanical loading increasesbone formation primarily at sites where there are high physical strainsand stresses in response to normal physical activities and to a muchless extent at sites where there are less physical strains and stressesimposed by normal physical activities. Thus, it is mechanical loading islikely to enhance the amount of endosteal/trabecular bone formed at agiven skeletal site in response to osteogenic agents (e.g., of FGF-2 orother bone growth promoting growth factors). Accordingly, the strategydescribed herein can be employed in combination with exercise, physicaltherapy and/or vibrational treatment modalities (e.g., ultrasound) thatsimulate mechanical loading without exercise to increaseendosteal/trabecular bone formation at appropriate skeletal sites,leading to the maximally increase in bone strength. Moreover, thecombination of administering Sca-1⁺ cells that express an osteogenicgrowth factor and appropriate mechanical or vibrational loading regimenincreases endosteal cortical bone formation and cortical thickness,which, along with the increased endosteal trabecular bone formation,further increases bone strength.

To eliminate or reduce side effects of massive and rapid endosteal boneformation, such as a reduction in medullary hematopoiesis due to theloss of marrow cavity, and increased extramedullary hematopoiesis withinthe spleen and liver resulting in enlargement of these two organs, thetreatment dose of serum FGF-2 should be maintained at a level of lessthan about 500 pg/ml (e.g., at less than about 200 pg/ml).

To reduce the risk of hypocalcemia, osteomalcia, and secondaryhyperparathyroidism in response to the large increase in bone formation,and eliminate secondary hyperparathyroidism, FGF-2 dosage can becontrolled and sufficient calcium and vitamin D supplementations can beadministered to subjects undergoing this osteogenic regimen.

Since all of the adverse effects can be avoided by limiting the extentof the increase in bone formation and/or controlling the dose of growthfactor. One way to control the dose is to regulate the expression of themodified FGF-2 transgene in the engrafted, transduced Sca-1⁺ cells. Inthis regard, the expression of the modified FGF-2 transgene in these MLVvectors was driven by the powerful constitutive CMV viral promoter. Aregulatable promoter can therefore be used to drive transgene expressionto control the amounts of FGF-2 protein produced and secreted byengrafted, transduced cells.

For example, a tet-on regulatable promoter can be used to obtainconsistent levels of FGF-2 that do not result in significant sideeffects. Briefly, two sets of MLV-based vectors have been constructedwith a tet-regulatable promoter [pY.tet.eGFP (in forward and reverseorientation) and pY.tet.FGF2*) expressing eGFP and the modified FGF-2gene, respectively (FIG. 7A). Murine Sca-1⁺, rat skin fibroblasts, andrat marrow stromal cells were transduced with pY.eGFP, pY.FGF2*,pY.tet.eGFP, as well as pY.tet.FGF2*, and the production of GFP protein(by FACS) and secretion of FGF-2 protein (by ELISA) in each CM weremeasured with or without doxycycline. With the GFP production as thetransgene production by the transduced cells, doxycycline produced adose-dependent production with the maximal production seen with 1 μg/mldoxycline (FIG. 7B), which was as high as or more than cells transducedwith the MLV vector without the tet promoter. The transduced cellsproduced only basal amounts (<2 ng/ml) of FGF-2 per 24 hrs withoutdoxycycline as compared to 48 ng/ml without doxycycline without the tetpromoter. With the addition of 1 μg.ml doxycycline, the secretion ofFGF-2 protein into the CMs by the transduced cells were increased bymore than 30-fold to above levels observed with the non-regulatablepromoter (68 ng/ml vs. 40 ng/ml). This tet-on promoter was just aseffective in Sca-1⁺ cells as in rat skin fibroblasts or in rat marrowstromal cells. Moreover, the tet-on promoter was effective in the senseor the reverse orientation (Table 13). Consequently, thistetracycline-inducible promoter system can be used to regulate FGF-2expression in Sca-1⁺ cells.

Thus, precisely controlling the secretion of FGF-2 by using aregulatable promoter system, such as the tet-on system, is useful toeliminate or reduce these undesirable side effects. Additionally, theuse of a regulatable promoter system to drive expression of the modifiedFGF-2 gene also allows stopping the treatment when an increase inendosteal bone formation is no longer needed.

With the exception of the avoidable side effects that appeared to beassociated with the robust bone formation and the high dose used, theosteogenic therapy described herein did not produce other significantharmful side effects. The treatment did not significantly affect growth,nor was it harmful to various vital organs.

The safety issue concerning insertion mutagenesis can be avoided byusing plasmid vectors. Two major problems have prevented the generalizeduse of plasmid vectors to deliver therapeutic agents: 1) lowtransfection efficiency, and 2) random insertion. However, recentimproved transfection reagents have significantly improved thetransfection efficiency of plasmid vectors. For example, the use ofnucleofection reagent yielded up to 90% transfection efficiency ofplasmid vector in mesenchymal stem cells (Lorenz et al., Biotechnol.Lett. 26:1589-1592, 2004). To address the issue of random insertion, asingle plasmid “Sleeping Beauty” transposon-based plasmid vector systemhas been developed, which yields stable integration of plasmid vectorprimarily at TA-dinucleotide sites (Harris et al., Anal. Biochem.:310:15-26, 2002). A nuclear localization sequence (NLS) has beenincluded in the “Sleeping Beauty” vector to promote its nucleartransport. This plasmid system can include a constitutive or regulatablepromoter (e.g., the tet-on promoter) depending on levels achievedfollowing transfection.

Although the Sca-1⁺ enriched cell population can favorably be used asthe target donor cell population in this system, this cell population isvery heterogenous and contains a number of different cell types inaddition to hematopoietic stem cells. Additional improvements can beobtained by using hematopoietic stem cells. Stem cells are pluripotentcells, which have the ability of self-renewal, engraft, expand, anddifferentiate into any cells, including cell of osteoblast lineage.Consequently, the use of hematopoietic stem cells, instead ofheterogeneous Sca-1⁺ enriched cell population, is likely to increase theengraftment efficiency as well as the osteogenic potentials. There isevidence that transplantation of a single hematopoietic stem cell into alethally irradiated animal is sufficient to repopulate the entirehematopoietic cell lineages. Consequently, a substantial reduction inthe number of cells used in the transplantation can be achieved by usinghematopoietic stem cells. In addition, stem cells are primitive cellsthat are not immunogenic, greatly reducing the incidence ofimmunorejection, graft-versus-host disease, and other adverse immuneresponses.

This discovery has great clinical significance and is extremely relevantto treatment of osteoporosis and related musculoskeletal diseases inthat a large portion of patients with osteoporosis is elderly.Frequently, osteoporosis is not diagnosed until the disease hasprogressed to a relatively advanced stage, and bone mass has beensignificantly reduced and, in some cases, multiple fractures havealready occurred. These patients require a therapy that can rapidly putback relatively large amounts of trabecular bones on their spines andhips within a relatively short time to avoid additional fractures.Accordingly, this osteogenic strategy is ideal for such subjects. Moreimportantly, this treatment strategy, is not only applicable to elderlyosteoporotic patients, but is also applicable to all patients withosteoporosis and related musculoskeletal diseases regardless of age orgender. Moreover, emerging studies suggest that hematopoietic(Schuldiner et al., Proc. Natl. Acad. Sci. USA 97:11307-11312, 2000) andmesenchymal (Spangrude et al., Science 241:58-62, 1988) stem cells hometo sites of tissue injury. Accordingly, this approach is useful for abroad range of applications including wound healing, e.g., fracturerepair.

Example 4 Sca-1⁺ Hematopoietic Progenitor Cell-Based Systemic GeneTherapy in Old Adult Animals is as Effective as that in Young AdultAnimals

Materials and Methods

Transplantation Bone Marrow Sca-1⁺ Cells

Bone marrow Sca-1⁺ cells were isolated from TgN-GFP mice and transducedwith MLV-based vectors as described in Example 3. The transduced cellswere used in the transplantation experiment within 12-24 hrs aftertransduction. Two weeks before and 2 weeks after the irradiationprocedure, One year-old (old adult) and 2-months-old (young adult)W⁴¹/W⁴¹ recipient mice were fed sterile food and autoclaved, acidifiedwater (pH 2.0-2.5) containing antibodies (50 mg/L neomycin sulfate and13 mg/L polymixin B sulfate) two weeks before and two weeks after thetotal body irradiation of a sublethal dose of 500 cGy at a rate of 80cGy per minute as described in Example 3. Half of the mice from the oldadult and the young adult groups received transplantation of 400,000TgN-GFP Sca-1⁺ cells transduced with the pY-βgal marker gene vector, andthe other half from each group received transplantation of 400,000TgN-GFP Sca-1⁺ cells transduced with the MLV.FGF2* vector as describedin Example 3. Mice were bled at 6, 8, 12, 14 weeks for analysis ofengraftment (FACS) and serum collected for biochemical marker assays andstored at −70° C. At 14 weeks post-transplantation, mice were euthanizedand femurs and tibia were collected for marrow, pQCT and bone histologyanalyses.

Other methods are described in details in Example 3. Non-parametricstatistics were used to determine statistical significance of parametersbecause serum FGF-2 levels in the recipient mice did not follow Gausiandistribution.

Results

Old Adult Mice Showed Similar Engraftment Efficiency as Young Adult Mice

To evaluate whether older adult mice have reduced engraftment efficiencyfor Sca-1⁺ cells, the engraftment efficiency of the transplanted GFPexpressing donor Sca-1⁺ cells in the older adult mice (1 year-old) wasdetermined and compared to that of the younger adult mice (2-month-old)by measuring GFP expression in the peripheral blood and in bone marrowcells in each recipient mice with FACS. Table 14 shows the chimericlevels in the peripheral blood of the 4 test groups. The engraftmentlevels in both the old and young adult group were relatively high(between 70-90%) at each time point. Regardless of the type oftransduced donor cells, the engraftment levels of the old adult groupwere similar to those of the young adult group. ANOVA analysis indicatesthat there was no significant difference in chimeric levels among the 4test groups.

TABLE 14 Comparison of the engraftment efficiency in the 1-year-old andthe 2-months-old adult recipient mice (mean ± S.D.) Group Treatment n 6weeks 8 weeks 10 weeks 12 weeks 14 weeks Old β-gal 9 77.66 ± 7.05 80.98± 7.23 82.45 ± 6.56 86.89 ± 5.17 82.77 ± 8.63 Young β-gal 10 75.86 ±4.19 78.65 ± 3.75 83.75 ± 3.61 89.88 ± 3.42 83.19 ± 6.10 Old FGF2* 10 71.25 ± 14.38 76.93 ± 7.22  77.56 ± 14.00 87.07 ± 5.92 84.42 ± 7.67 OldFGF2* 9 76.11 ± 5.50 83.12 ± 5.61 83.63 ± 5.29 87.04 ± 6.08 83.67 ± 5.10ANOVA N.S. N.S. N.S. N.S. N.S.

As previously reported, the levels of chimera in the bone marrow cellsof each recipient mouse were significantly lower than those in theperipheral blood. At the time of euthanasia, the level of the chimera inthe old adult group transplanted with the MLV.β-gal- and theMLV.FGF2*-transduced Sca-1⁺ donor cells was 20.47±5.19% and 23.66±7.55%,respectively. The level of the chimera in the young adult grouptransplanted with the two vectors was 22.35±5.14% and 21.18%±6.30%,respectively. There was no significant difference in the chimera levelsin the bone marrow among the four test groups. Thus, these findings areconsistent with the general belief that the engraftment of donor cellsin the peripheral blood is a good indicator of the true bone marrowengraftment. These findings together indicate that the engraftment ofthe donor Sca-1⁺ cells in the old adult mice is just as efficient asthat in the young adult mice, and confirm the findings shown in Example3 that the expression of the modified FGF-2 did not modify theengraftment efficiency in either the old or the young adult animals.

Comparison of FGF2 Transgene Expression Between Old Adult Mice and YoungAdult Mice.

In order for this systemic Sca-1⁺ cell based FGF-2 gene therapy to beeffective in old adult mice, the old adult recipient mice should be atleast just as effective in expressing the FGF2 transgene as in youngadult recipient. Accordingly, the serum FGF-2 level in the old adultmice after engraftment (at 8- and 10-weeks after transplantation) wasmeasured with a commercial FGF-2 ELISA kit as an indicator of theability of the old adult mice to express the FGF2 transgene. FIG. 8Areveals that, at each of the test time points (8- or 10-weekspost-transplantation), the old adult recipient mice receiving Sca-1⁺cells transduced with the MLV. FGF2* vector showed a significantlyhigher serum FGF2 levels than those young adult recipient mice receivingthese FGF-2 expressing cells. The serum FGF-2 level in old adult micetransplanted with Sca-1⁺ cells transduced with the MLV.β-gal controlvector was also not significantly different from that in young adultmice transplanted with the untransduced control cells. Because there wasno significant difference in the engraftment, the larger increase inserum FGF-2 in the old FGF-2 group compared to that in the young FGF-2group was due to the enhanced ability of the engrafted cells to expressthe FGF2 protein in the old adult mice compared to young adult mice. Asreported in Example 3, the variation in serum FGF-2 levels in micetransplanted with the FGF-2 expressing Sca-1⁺ cells was relatively largein the young adult mice. Thus, the relatively large variation in serumFGF-2 levels was unrelated to the age of the recipient mice.

To confirm that serum FGF-2 levels in these recipient mice areacceptable indices for FGF-2 transgene expression level, the modifiedFGF-2 mRNA levels in the bone marrow of each recipient mouse were alsomeasure by real-time PCR. The bone marrow FGF-2 mRNA levels correlatedlinearly and strongly with serum FGF-2 levels at each time point(r=0.86, p<0.0.0001, at 8 weeks post-transplantation; and r=0.69,p<0.0.0001, at 10-weeks time point). Thus, serum FGF-2 levels aresuitable indices for the FGF-2 transgene expression in these recipientmice. Importantly, these data indicate that the older mice did not havea reduced capacity to express the FGF-2 transgene.

Transplantation of Modified FGF-2 Expressing Sca-1⁺ Cells EffectivelyInduced Endosteal Bone Formation in Recipient Mice

Serum and bone alkaline phosphatase activity (ALP) and pQCT parametersin the recipient mice were measured at the time of euthanasia (14 weeks)as indices for bone formation response assessments. Similar to the serumFGF-2 levels, the variations in serum and bone ALP activities(normalized against protein or dried bone weight) in recipient micetransplanted with FGF-2 expressing Sca-1⁺ cells were also large. As aresult, none of the differences in serum and bone ALP activity in thisexperiment reached the statistically significant level by the parametricstatistical tests. However, non-parametric statistical analysisindicates that there was a significant (p<0.03) increase in tibialALP/protein in FGF-2 expressing mice compared with β-gal expressing mice(for both old and young adults).

The total cortical and trabecular BMD as well as periosteal andendosteal bone parameters were measured in these mice by pQCT. Withrespect to trabecular bone mineral density (BMD), as expected, oldermice have significantly lower trabecular BMD than younger mice. With thenon-parametric Krusal-Wallis test, the FGF-2 mice showed a significantlyincrease in trabecular BMD at their corresponding age group (p=0.0052),indicating that the FGF2 treatment effectively increased trabecular BMDin both young and old mice. Correlation analyses indicate that there wasa significant and positive correlation (r=0.34, p<0.05) between serumFGF-2 and trabecular bone mineral density (BMD) of these mice. Thesefindings support the contention that transgenic expression of FGF-2 inthe bone marrow space by the Sca-1⁺ cell-based systematic gene therapycan effectively induce endosteal bone formation in old as well as youngadult mice.

To confirm an endosteal bone formation response in the old adult mice,bone histomorphometric analysis was performed on the femurs of old andyoung recipient mice transplanted with either β-gal expressing or FGF-2expressing Sca-1⁺ cells. Briefly, recipient femurs were cut in half. Thedistal bone half was demineralized and embedded in paraffin while theproximal half was embedded in methylmethacrylate. Longitudinal sectionswere prepared from the paraffin-embedded bones were stained with H & Efor tissue structure or Mallory's trichrome stain for collagen,respectively. Longitudinal sections were prepared from themethylmethacrylate embedded bones and the sections were stained withGoldner's stain for mineralization. To demonstrate the dose-dependenteffect, histology results of a representative mouse expressing moderateand a representative mouse expressing high levels of FGF-2 as well as amouse receiving β-gal were included.

Transplantation of FGF-2 expressing Sca-1⁺ cells resulted in massivebone formation in young as well as old adult mice as indicated by theMallory's staining. Furthermore, the massive increase in bone formationwas seen at diaphyses, metaphyses, as well as epiphyses of the femur,consistent with the premise that the treatment caused systemic boneformation along the endosteal bone surfaces. There is evidence that theenhanced endosteal/trabecular bone formation in the older mice was alsodose-dependent, since the increase in bone formation was much higher inmice with high serum FGF-2 levels compared to those with moderate of lowserum FGF2.

The total bone area of the bone sections were also determined, and isshown in FIG. 8B. The FGF2* treatment significantly increased the bonearea in both the young and the old animals, confirming the osteogeniceffect of this Sca-1⁺ cell-based systemic gene therapy. Consistent withthe higher serum FGF2 levels in the older mice, the bone area in theFGF2*-treated older animals was also much bigger than that of theFGF2*-treated younger mice. These bone histological evidence clearlydemonstrated that this Sca-1⁺ cell-based systematic therapy is just aseffective (if not more so) in the old adult mice as that in the youngadult mice.

As shown in Example 3, the rapid and massive increase in bone formationin recipient mice that showed high levels of serum FGF2 can causecalcium deficiency, osteomalacia, and secondary hyperparathyroidism.This is a concern especially with respect to the older animals, since ithas been well established that calcium absorption is not as efficient inthe elderly as that in young adults. To determine if the older micetreated with FGF2 expressing Sca-1⁺ cells did not develop a more severecalcium deficiency problem than the young mice receiving theFGF2-expressing Sca-1⁺ donor cells, the serum albumin-adjusted calciumlevels of the young and old recipient mice were determined (as asurrogate measure of ionized calcium levels) and compared (Table 15).Two-way ANOVA indicates that the FGF-2 treatment significantly(p<0.0001) increased total as well as albumin-adjusted serum calciumlevels in both young and old recipient mice. Tukey's HSD a prioritesting shows that young β-gal group was significantly (p<0.05) higherthan the old mice transplanted with β-gal expressing donor cells. Boththe young and old FGF2 group had a significantly (p<0.01 for each)higher total and adjust calcium levels than their corresponding β-galgroup. These findings confirm that the high expression levels of FGF-2did not produce a bigger problem in causing calcium deficiency in theolder animals than that in the young adult mice.

TABLE 15 Comparison of the albumin-adjusted serum calcium levels in theold recipient mice transplanted with Sca-1⁺ cells expressing β-gal orthe modified FGF-2. total calcium and albumin was measured at endpoint(14 weeks). Corrected calcium was calculated using the followingformula: Corrected calcium = total calcium + 0.8 (4.5 − albumin). (mean± SD, n = 9 or 10 per group). Age group Treatment Serum albumin TotalCalcium Adjusted Calcium Old β-gal 4.0 ± 0.1 10.4 ± 0.3 10.8 ± 0.3 OldFGF2* 3.9 ± 0.3 11.2 ± 0.3 11.7 ± 0.3 Young β-gal 4.0 ± 0.1 10.9 ± 0.511.3 ± 0.6 Young FGF2* 3.9 ± 0.2 11.4 ± 0.4 11.9 ± 0.4 ANOVA, p value0.24 <0.0001 <0.0001

To assess if osteomalacia occurred, bone sections of the old recipientmice expressing low (49 pg/ml), moderate (840.4 pg/ml) or high (2,933pg/ml) serum FGF-2 at the metaphyses and epiphyses sites were alsostained with the Goldner's stain to distinguish mineralized andunmineralized bone matrix. Similar to the results observed in younganimals as discussed in Example 3, the old FGF2*-treated mouseexpressing very high levels of FGF2 exhibited a relatively large amountof newly formed, unmineralized bone matrix overlaying the mineralizedtrabeculae. Bone formation was so massive and so rapid that it ispossible that insufficient amounts of calcium were present to mineralizethe newly formed bone in those mice with very high serum FGF2 levels.However, compared to the results shown in Example 3, the amounts ofnewly formed, un-mineralized bone matrix in the old adult mice thatexpressed high serum levels of FGF-2 did not appear to be more thanthose in the young adult mice with similar serum FGF-2 levels,demonstrating that suboptimal calcium levels were not any more severe inthe older animals than in the younger mice.

In summary, the findings shown in this Example has clearly indicatedthat this Sca-1⁺ cell-based systemic gene therapy is as effective, ifnot more so, in the older mice. Consequently, these findings support thecontention that this therapy is well suited for the elderly osteoporoticpatients, who require a therapy that can put back relatively largeamounts of trabecular bones at endosteal bone surfaces within theirspines and hips within a relatively short time to avoid furtherfractures.

Example 5 Unmodified FGF-2 can be Used to Promote Endosteal TrabecularBone Formation

The modified FGF2 transgene, rather than unmodified FGF2 gene, was usedin the other Examples was based on the facts that 1) FGF2 lacks aclassifical secretion signal sequence and its extracellular secretion ismediated by an energy-dependent non-ER/golgi pathway (Mignatti et al., JCell Physiol 151:81-93, 1992; Florkiewicz et al., J Cell Physiol162:388-399, 1995; Dahl et al., Biochemistry 39:14877-14883, 2000),which is highly inefficient, leading to very poor secretion rate of FGF2in mammalian cells (Moscatelli et al., J Cell Physiol 129:273-276,1986); and 2) FGF2 protein can form various disulfide forms, which canresult in significant loss of biological activities and proteinstability (Iwane et al., Biochem Biophys Res Commun 146:470-477, 1987).However, the efficiency of Sca-1⁺ cells to secrete the unmodified FGF2has not been evaluated. This Example evaluated whether the engraftedSca-1⁺ cells secrete sufficient amounts of an unmodified FGF2 transgeneby this systemic gene transfer protocol. For comparison, Sca-1⁺ cellstransduced with a MLV vector expressing a modified FGF2 with only theadded BMP2/4 pro-peptide but without the cysteine mutations(MLV.proFGF2) was also included for testing.

Materials and Methods

Six-months-old W⁴¹/W⁴¹ recipients were divided into 4 groups and weretransplanted with 500,000 Sca-1+ cells from GFP transgenic micetransduced to express β-gal, unmodified WT FGF2 transgene, modified FGF2with only the BMP2/4 propeptide gene or the FGF2* mutant gene asdescribed in Example 3. Blood samples were obtained for analysis ofengraftment (by FACS analyses of GFP expression) and for measurements ofserum FGF2 levels at 6, 8, 10, and 14 weeks post-transplantation. Serumbiochemical markers and calcium were also measured.

Results

Assessment of engraftment was performed by measuring the relativepercentage of GFP expressing cells in the peripheral blood (by FACS) ofeach recipient mouse. Table 16 shows that there was no significantdifference in the engraftment level among each of the four test group.

TABLE 16 Relative engraftment levels in mice transplanted with Sca-1⁺cells expressing wild type (WT-FGF2), modified FGF2 with only the BMP2/4propeptide (Pro. FGF2), and the modified FGF2 (FGF2*) at 6 weeks, 8weeks, or 10 weeks post-transplantation (mean ± SD). Group N 6 weeks 8weeks 10 weeks β-gal 5 79.89 ± 5.21 87.12 ± 1.95 84.91 ± 2.55 WT-FGF2 1080.34 ± 6.63 87.25 ± 3.02 82.95 ± 3.97 Pro.FGF2 10 80.65 ± 6.47 88.83 ±2.38 83.76 ± 4.73 FGF2* 10 78.49 ± 6.70 85.78 ± 4.87 79.97 ± 6.32 ANOVAN.S. N.S. N.S.

To confirm engraftment, the RNA levels of the FGF2 transgene in the bonemarrow cells of each of the recipient mice were measured by real-timePCR with a set of specific primers at the end-point of the experiment(10 weeks post-transplantation). The results were shown as relative foldchanges in relationship to the housekeeping gene, cyclophilin, by theΔCT method. Table 17 confirms engraftment.

TABLE 17 Relative FGF2 mRNA in bone marrow cells of recipient mice(relativefold changes with respect to cyclophilin housekeeping gene).Relative levels of FGF-2 mRNA/ Group N Cyclophilin mRNA β-gal 7 0.00 ±0.00 WT-FGF2 10 0.0056 ± 0.0165 Pro-FGF2 9 0.0070 ± 0.0104 FGF2* 90.0070 ± 0.0104

To measure the FGF2 transgene expression, serum FGF2 levels were assayedat 6 weeks post-transplantation using a FGF2 commercial ELISA kit.Surprisingly, the serum FGF2 levels in the unmodified FGF2 group wereincreased nearly 4-fold over the β-gal controls (FIG. 9), suggestingthat Sca-1⁺ cells transduced with the unmodified FGF2 secretesubstantial amounts of FGF2 protein. Nevertheless, the secretion levelswere still significantly lower (>2-fold) than that of mice in themodified FGF2 group (487±869 pg/mL, p<0.001). The wide variation inmeasurement was observed in both the modified and unmodified FGF2groups, suggesting it is not a result of the gene modification.Moreover, linear regression analysis revealed that the serum FGF-2levels in these recipient mice correlated significantly with the FGF-2transgene mRNA levels in their bone marrow cells (r=0.44, p=0.0099),indicating that the engrafted cells indeed expressed the FGF2 transgenemRNA.

Serum total calcium, albumin-adjusted calcium, and serum bone formationbiochemical markers were also measured in these recipient mice. Table 18shows that in this Example, none of the groups showed evidence forcalcium deficiency. ANOVA analysis revealed that the serum ALP levels inthese recipient mice were not significantly different, presumably due tolarge variation. Conversely, recipient mice of the FGF2* group showedsignificant increases in serum CTX and osteocalcin. However, thesebiochemical markers in the WT FGF2 and the Pro.FGF2 groups were notsignificantly different from the β-gal control group.

TABLE 19 Serum calcium levels and bone formation biochemical markerslevels of recipient mice. Results are shown as mean ± SD. Albumin-adjusted Serum Serum Serum Group N Total Ca Albumin Calcium ALP CTXOsteocalcin β-gal 5 10.24 ± 0.33 3.88 ± 0.16 10.74 ± 0.22 95.00 ± 9.902.14 ± 1.67 40.36 ± 7.23 WT- 10 10.34 ± 0.23 4.02 ± 0.19 10.72 ± 0.1894.60 ± 14.07 1.61 ± 0.95 33.16 ± 8.37 FGF2 Pro.FGF2 9 10.04 ± 0.16 3.89± 0.21 10.53 ± 0.19 91.89 ± 11.58 3.02 ± 1.78 51.60 ± 16.29 FGF2* 1010.21 ± 0.39 3.96 ± 0.20 10.64 ± 0.35 97.10 ± 18.74 3.66 ± 1.75 54.26 ±13.22 ANOVA N.S. N.S. N.S. N.S. 0.035 0.002 Corrected calcium wascalculated using the following formula: Corrected calcium = totalcalcium + 0.8 (4.5 − albumin)

Bone histomorphometric analyses indicate that only mice from the FGF2*group show large increase in endosteal/trabecular bone formation, whilemice of the WT FGF2 group did not show consistent histomorphometricevidence of an increased endosteal bone formation (data not shown).Quantitation of bone areas confirm that recipient mice of the FGF2*expressing cells, but not those of WT FGF2 expressing mice showed anincrease in percent bone area (6.97±4.71% for the β-gal group;6.78±3.75% for the WT-FGF2 group, and 16.02±7.51% for the FGF2* group).Linear regression indicates that the percent bone area correlatedsignificantly with serum FGF2 levels (r=0.79, p<0.0007), a finding isconsistent with the interpretation that the increase bone mass in theserecipient mice was due to FGF2 expression.

In summary, transplantation of Sca-1⁺ cells in the recipient mice led toan increase in serum FGF2 levels. However, in spite of the higherexpression of FGF2 in the wild type FGF2 gene group compared tocontrols, there was no evidence for a consistent increase in endostealtrabecular bone formation. There are at least two possibleexplanations: 1) the amounts of FGF2 produced by the Sca-1⁺ cellstransduced with the MLV.WT-FGF2 vector is not be sufficient toconsistently produce an osteogenic effect, and/or 2) the modified FGF2gene possesses more potent osteogenic functions than the WT FGF2.

Example 6 Transplantation of Human CD34⁺/Lin⁻ HematopoieticStem/Progenitor Cells that Express a Modified FGF-2 Transgene PromotesEndosteal/Trabecular Bone Formation

This example illustrates that human hematopoietic stem/progenitor cellsare suitable cell vehicle source and that this hematopoieticstem/progenitor cell-based systemic gene therapy protocol is suitablefor humans. In this Example, the effectiveness of human hematopoieticstem/progenitor cells as a cell vehicle is assessed in athymic “nude”mice in order to avoid an immune response against the transplanted humancells. CD34 is a cell surface glycoprotein (also known as mucosialin)that is expressed on human hematopoietic stem cells, but not on matureblood cells. CD34 is a well recognized cell surface marker of humanhematopoietic stem cells and progenitor cells. Human CD34⁺ (e.g., CD34⁺,lin⁻, AC133⁺, CD45⁻, CXCR4⁺) cells are hematopoietic stem/progenitorcells used as the ex vivo cell vehicle to express the modified FGF-2transgene (FGF-2*) to induce endosteal trabecular bone.

Materials and Methods

Animals

Recipient mice for transplantation of human cells to produce a workingmodel of human hematopoietic stem cell transplantation are athymic“nude” mice. These mice lack a functional thymus and are an ideal animalmodel for studying allografts and xenografts. These mice are maintainedin a complete sterile environment.

Transplantation Strategy

The transplantation strategy is very similar to that described inExample 3, with the exception that transduced human CD34⁺ cells are usedas the donor cells. Highly purified human CD34⁺ cells (available fromcommercial sources, such as Cambrex Bio Science Walkersville, Inc) canbe expanded in culture by maintaining the stem cells in serum-freemedium containing stem cell factor, thrombopoietin, IGF-2, FGF-2, andangiopoietin 2. Transduction of the pY.eGFP (for assessment ofengraftment), pY.FGF2*, and pY.tet.FGF2* vector is performed asdescribed above. After appropriate conditioning, each nude mousereceives 300,000 transduced CD34⁺ cells as described in Example 3.Recipient mice that receive pY.tet.FGF2* transduced cells are treatedwith an effective dose of doxycycline in their drinking water toinitiate transgene expression. Blood is drawn at various time points.Engraftment is assessed by detecting GFP production in peripheral cellsof the transduced mouse. Bone formation effects are analyzed by BMDmeasurements using pQCT, serum biochemical bone formation markers, andhistological evaluations of bone formation as described in Example 3.

Results

Transplantation of human CD34⁺ cells in athymic nude mice is predictedto increase endosteal trabecular bone formation. The increase intrabecular bone formation is proportional to the serum FGF-2 levels inthese mice. The induced trabecular bones are of normal quality andmicrostructure. The increase in endosteal bone formation leads to anincrease in overall bone strength. The serum FGF-2 levels and therebythe amounts of new bone formation in nude mice receiving thepY.tet.FGF2* transduced cells is regulatable by adjusting thedoxycycline dose in the drinking water.

Example 7 Transplantation of Hematopoietic Stem/Progenitor Cells thatExpress Wnt1 Promotes Endosteal/Trabecular Bone Formation

Bone growth factor genes that have the ability to expand the stem cellpopulation and to stimulate bone formation are able to promote endostealtrabecular bone formation when delivered using transduced hematopoieticstem and/or progenitor cells.

Materials and Methods

Construction of pY.Wnt1a Vector

The full length Wnt1 cDNA was cloned from the mouse Wnt1 gene (obtainedfrom the ATCC). The resulting PCR product was subcloned into theMLV-based pY vector backbone to generate the pY-Wnt1 vector. SeveralpY.Wnt1 clones were randomly selected and sequenced to confirm theidentity pY.Wnt1 vector.

Animals

As described in Example 3, W⁴¹/W⁴¹ mice are used as the recipient mice,and Sca-1⁺ cells of the GFP expressing transgenic mice are used as donorcells to demonstrate efficacy of osteogenic growth factors other thanFGF-2 in the induction of bone formation.

Transplantation Strategy

Sca-1⁺ cells are transduced with either the pY.Wnt1 vector or thepY.β-gal control vector. For comparison, the same Sca-1⁺ cells are alsotransduced with the pY.FGF2* vector. The transplantation procedure isidentical to that described in Example 3. Blood is drawn at various timepoints. Engraftment is assessed by GFP production in peripheral cells ofthe transduced mouse. Bone formation effects can be analyzed by BMDmeasurements using pQCT, serum biochemical bone formation markers, andhistological evaluations of bone formation as described in Example 3.

Results

The MLV-based vectors expressing the murine Wnt1a was generated andHT1080 cells were transduced with the vector. The relative levels ofWnt1 protein in the CMs and cell lysates, 72 hours after thetransduction, were identified by Western immunoblots. HT1080 cellstransduced with pY.β-gal showed undetectable levels of Wnt1 protein inCMs or in cell lysates. In contrast, there were high levels ofimmunoreactive Wnt1 proteins in both the CM and cell lysate of thepY.Wnt1 transduced cells, indicating that the pY.Wnt1 vector was capableof inducing Wnt1 protein synthesis.

To confirm that growth factors other than FGF-2 that satisfy thecriteria of 1) promoting self-renewal of hematopoietic stem cells, and2) inducing bone formation induce the formation of bone in vivo, thepY.Wnt1 vector, along with the pY.β-gal and pY.FGF2* vectors, aretransduced into Sca-1⁺ cells as described in Example 3. Blood is drawnat various time points. Engraftment is assessed by the GFP production inperipheral cells of the transduced mouse. Bone formation effects can beanalyzed by BMD measurements using pQCT, serum biochemical boneformation markers, and histological evaluations of bone formation asdescribed in Example 3.

Results

Transplantation of pY.Wnt1 transduced Sca-1⁺ cells is predicted toproduce massive new trabecular bone formation at endosteal bone sites.The bone formation is proportional to the serum levels of Wnt1.

Example 8 Transplantation of Hematopoietic Stem Cells and/or ProgenitorCells that Express FGF-2 Promotes Bone Formation is Broadly Applicable

The effectiveness of hematopoietic stem/progenitor cell transplantationto induce bone formation in different animals can be confirmed using alarger, non-rodent animal, such as the beagle dog. Effective delivery ofosteogenic growth factors using hematopoietic stem/progenitor cells toinduce bone formation is not limited to small rodents, but also appliesto higher mammals, such as cats, dogs, domesticated livestock (such assheep, goats, pigs and cows), horses and captured wild-animals, as wellas non-human primates and humans. In this experimental example, dogs areselected as the recipient because the bone metabolism of dogs closelyresembles that of humans.

Materials and Methods

Animals

Eight month old normal beagle dogs weighing 12 kg are used as thetransplantation recipient. To avoid the need for immunosuppressivetherapy, autologous marrow transplantation is used.

Transplantation Strategy

Before the experiment, the dogs receive baseline measurements of bone(including a bone density test and blood and urine tests for bonemetabolism) and hematology assessment. To prevent the calcium deficiencyrelated to massive new bone formation, the dogs receive 200 mg ofcalcium supplementation along with 500 international units of vitamin Dprior to and during the period of evaluation. In some cases,erythropoietin therapy is administered before the transplantationprocedure to enhance bone formation in sites throughout the skeleton,including fatty marrow sites.

The recipient dogs receive five daily injections of GCSF (canine, fromAmgen) at a dose of 10 mg per kilogram to enhance the number of bonemarrow progenitor cells in the harvested marrow; this treatment alsoresults in more rapid engraftment. The hematopoietic stem/progenitorcells are harvested by apheresis from the dog's blood. The leukocytefraction is frozen until approximately 24 hours before transplantation.The harvested leukocytes are enriched for CD34⁺, lin⁻ progenitor cells.Briefly, the leukocytes are labeled with biotinylated monoclonalantibody 1H6 (immunoglobulin G1 anti-canine CD 34) 40 μg per milliliterat 4° C. for 30 minutes. The cells are incubated withstreptavidin-conjugated microbeads for 30 minutes and 4° C.; and thenseparated by using an immuno magnetic column technique (MiltenyiBiotech, Auburn Calif.). Depending upon the number of stem cellsharvested by leukapheresis, the hematopoietic stem/precursor cells canbe expanded by culturing the stem cells in serum-free medium containingstem cell factor, thrombopoietin, IGF-2, FGF-2, and angiopoietin 2.Transduction can be accomplished with the MLV.tet.FGF2* vector, whichcontains a tet-on regulatable promoter that allows regulation of thegene therapy.

Prior to transplantation, the recipient dogs are conditioned withnonmyeloablative total body irradiation of 200 to 300 cGy at 7 cGy/Minor Fludabarine (500 mg per meter squared) at five separate doses withoutthe radiation. Four to 24 hours after the conditioning, the dogs receivethe engineered hematopoietic bone marrow stem/precursor cells fortransplantation by IV infusion of 4.0×10⁸ cells per kilogram.Tetracycline (or an analog thereof) is administered orally to initiatethe transgene expression. Transgene expression is maintained as long asthe dogs are given the tetracycline. Once the tetracycline is stopped,expression of the introduced osteogenic growth factor is silenced. Inthis way, the dose of the gene therapy can be regulated.

The standard follow-up for marrow transplantation is provided, includingmonitoring of the peripheral white blood count. In addition, therecipients are monitored for calcium deficiency. Evidence of calciumdeficiency includes low serum calcium and a serum PTH above the uppernormal limit. This can be preceded by very large increases in serumalkaline phosphatase and osteocalcin. When evidence of calciumdeficiency is detected, the tetracycline administration can bediscontinued in order to silence expression of the growth factor genefor the period sufficient to correct the calcium deficiency, after whichthe gene therapy can be reinitiated. Optionally, calcium intake can beincreased by 100 mg increments.

To enhance the effects of the osteogenic therapy, the dogs are trainedfor an exercise program. Such exercises are important to influence theamount and orientation of the new bone being deposited. During therapyas the bone density increases the dogs can be trained to increase theintensity of the exercise program.

Therapeutic efficacy is monitored primarily by bone density monitoring,which can be measured periodically during therapy. Blood samples canalso be obtained for measurements of biochemical markers of boneturnover. To confirm the extent and quality of bone formation, a bonebiopsy can be obtained and bone histology can be performed.

Results

Transplantation of hematopoietic stem cells that express osteogenicgrowth factors is predicted to result in increased bone formation inanimals other than rodents, including humans. The newly formed bone isnormal in microstructure and can be produced without inducing calciumdeficiency or osteomalacia. Typically, bone formation is concentrated tothe endosteal bone surfaces. The serum biochemical markers of boneformation (serum alkaline phosphatase and osteocalcin) increasesubstantially as compared to control subjects. Overall, it is predictedthat bone density in treated subjects is predicted to increase by 25% ormore compared to control subjects.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A recombinant nucleic acid encoding a modified fibroblastgrowth factor-2 (FGF-2) analog, wherein the nucleic acid comprises in a5′ to 3′ direction: a polynucleotide sequence that encodes a bonemorphogenetic protein 2/4 (BMP2/4) hybrid secretion signal sequence; anda polynucleotide sequence that encodes a mature human FGF-2 polypeptide,wherein the mature human FGF-2 polypeptide comprises: a mutation thatresults in the substitution of an alanine for a cysteine in amino acidposition 70 (C2) of FGF-2, and a mutation that results in thesubstitution of an asparagine for a cysteine in amino acid position 88(C3) of FGF-2.
 2. A modified fibroblast growth factor-2 (FGF-2) analogencoded by a nucleic acid; the nucleic acid comprises in a 5′ to 3′direction: a polynucleotide sequence that encodes a bone morphogeneticprotein 2/4 (BMP2/4) hybrid secretion signal sequence; and apolynucleotide sequence that encodes a mature human FGF-2 polypeptide,wherein the mature human FGF-2 polypeptide comprises: a mutation thatresults in the substitution of an alanine for a cysteine in amino acidposition 70 (C2) of FGF-2, and a mutation that results in thesubstitution of an asparagine for a cysteine in amino acid position 88(C3) of FGF-2.